Anti-huntingtin antibodies

Abstract

Antibodies that bind HTT proteins and fragments thereof are described. Compositions comprising these antibodies, as well as methods including therapeutic methods, are also described.

Description

[0001] This application claims priority to EP 22212053.7 filed on December 7, 2022 and GB 2305916.5 filed on April 21, 2023, the contents and elements of each of which are incorporated herein by reference for all purposes. Technical Field

[0002] The present invention relates to antibodies and fragments thereof capable of binding to Huntingtin (HTT), and particularly, but not exclusively, to improved therapeutic antibodies. Methods of using anti-HTT antibodies to treat neurological disorders are also described. Background Art

[0003] Huntington's disease (HD) is a neurodegenerative monogenic autosomal dominant disorder caused by a CAG expansion in exon 1 of the gene encoding the huntingtin protein (HTT). Despite its well-defined genetic origin, the molecular / cellular mechanisms underlying HD are complex. Current treatments focus on clinical manifestations (e.g., monoamine depletion agents, sulpiride / dopamine D2 receptor antagonists, and antidepressants) (Dash D and Mestre TA (2020) Therapeutic Update on Huntington's Disease: Symptomatic treatments and emerging disease modifying therapies Neurotherapeutics. 2020 Oct; 17(4): 1645-1659). These treatments alleviate some of the symptoms caused by HD but are unable to alter disease modification. Although oligonucleotide, gene, and cell therapies continue to advance in clinical development, significant obstacles remain, which may reflect delivery issues, the complexity of new modalities, the lack of distinction between the pathological / physiological functions of HTT, and patient acceptance of clinical trial designs. There remains a need for improved treatments for Huntington's disease.

[0004] Alzheimer's disease (AD) is a neurodegenerative disease that causes a progressive loss of brain cells. According to the 2016 World Alzheimer's Report (World Alzheimer Report 2016) (Comas-Herrera et al (2016) World Alzheimer Report 2016 www.alzint.org / resource / world-alzheimer-report-2016), 46.8 million people worldwide suffered from dementia in 2015, and this number is expected to reach 131.5 million by 2050. New treatments for Alzheimer's disease are being actively sought to modify the course of the disease. Current candidates targeting β-amyloid protein, Tau, and innate immunity in the brain have shown efficacy against pathological mechanisms in some cases in clinical trials, but have not yet demonstrated convincing disease modification in late-stage clinical trials. There remains a need for improved disease-modifying treatments for Alzheimer's disease (Golde TE (2022) Neurotherapeutics 19, 209-227).

[0005] The present invention has been devised in view of the above considerations. Summary of the Invention

[0006] The present invention relates to new and improved antibodies against HTT. By studying the immune response of individuals who showed resilience to neurodegeneration despite an increased risk of the disease and comparing it to progressive disease, the inventors identified clusters of related antibody heavy chains (VH) that converged in the recovering individuals. Target deconvolution showed that the convergent VHs could bind to HTT. The inventors also identified candidate antibodies derived from these identified VHs and used an unbiased approach of both antibody discovery and target identification to identify antibodies that are expected to slow or reverse neurodegeneration. In particular, representative heavy chains were paired with appropriate light chains and expressed in IgG1 format as antibodies referred to herein as ATL5331, ATL5334, and ATL5335. These antibodies were developed to further improve properties, not limited to improved binding potency, improved pharmacokinetic properties, reduced toxicity, and improved stability. These steps go beyond conventional optimization and required cohort diversity analysis, extensive testing, simultaneous investigation of multiple beneficial and mechanistic properties, and guided engineering to produce antibodies that do not exist in naturally occurring populations. These novel antibodies with improved properties include antibodies referred to herein as ATL5895, ATL5901, and ATL5667, and affinity-matured versions thereof. The antibodies described herein are expected to slow or reverse neurodegeneration by binding to mutant HTT (mHTT) and / or aggregated HTT, particularly its extracellular form.

[0007] In a first aspect, the present disclosure provides an isolated antibody or antibody fragment thereof that specifically binds to huntingtin (HTT) or a fragment thereof, the antibody comprising a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, and a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3), or a sequence comprising one or two substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3), wherein the substitutions are at positions selected from positions 95 and 97, wherein the substitutions are selected from Y97F and P95S, wherein position numbering is according to Kabat; iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ID NO: 4); v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and vi. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6) or an amino acid sequence comprising one, two, three, or four amino acid substitutions compared to GSYAGTANV (SEQ ID NO: 6), wherein the substitutions are at positions selected from the group consisting of: A92G, A95E, G89V, and Y91F, wherein the position numbering is according to Kabat.

[0008] Thus, an antibody may have the following HCDR1, HCDR2 and HCDR3:

[0009] ATL 5895, ATL_6194, ATL_6195, ATL_6374, ATL_6375, ATL_6376, ATL_6377, ATL_6378, ATL_6199, ATL_6200, ATL_6202, ATL_6203, ATL_6204 and / or ATL_6205,

[0010] and LCDR1, LCDR2, and LCDR3 below:

[0011] ATL 5895, ATL_6194, ATL_6195, ATL_6374, ATL_6375, ATL_6376, ATL_6377, ATL_6378, ATL_6199, ATL_6200, ATL_6202, ATL_6203, ATL_6204 and / or ATL_6205.

[0012] Embodiments of any aspect may have any one or more of the following optional features.

[0013] The isolated antibody or fragment thereof may have improved binding to a mutated and / or aggregated HTT protein compared to a non-mutated and / or non-aggregated HTT protein, wherein the relative binding to mutated and / or aggregated HTT and non-mutated and / or non-aggregated HTT is measured by determining the ratio of the EC50 value for an HTT protein, or a fragment thereof, comprising a 25Q repeat in exon 1 to the EC50 value for an HTT protein, or a fragment thereof, comprising a 48Q repeat in exon 1. The ratio of the EC50 for binding of the isolated antibody or fragment thereof to an HTT protein, or a fragment thereof, comprising a 25Q repeat in exon 1 to the EC50 for binding to an HTT protein, or a fragment thereof, comprising a 48Q repeat in exon 1 is at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2, as measured by sandwich ELISA. HTT or HTT fragments comprising 48Q repeats may comprise the sequence of SEQ ID NO: 44 or 46, and / or HTT or HTT fragments comprising 25Q repeats may comprise the sequence of SEQ ID NO: 43 or 45. Sandwich ELISAs may be performed as described herein (Examples, Materials and Methods).

[0014] In some embodiments, the antibody fragment comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3). In some such embodiments, the antibody or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6). In other such embodiments, the antibody or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence VSYGGTENV (SEQ ID NO: 162). In other such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence VSFAGTANV (SEQ ID NO: 160). In other such embodiments, the antibody or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence VSYAGTANV (SEQ ID NO: 161). Some examples of antibodies according to these embodiments include ATL_5895, ATL_6194, ATL_6195, ATL_6374, ATL_6375, ATL_6199, ATL_6200, ATL_6204, and ATL6205.

[0015] In some embodiments, the antibody or fragment comprises: a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence PPFYYYYGLDV (SEQ ID NO: 158); and a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 comprises the amino acid sequence VSYGGTENV (SEQ ID NO: 162). Some examples of antibodies according to these embodiments include ATL_6376 and ATL_6202.

[0016] In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence SPYYYYYGLDV (SWQ ID NO: 157). In some such embodiments, the antibody or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence VSYAGTANV (SEQ ID NO: 161). Some examples of antibodies according to these embodiments include ATL_6377 and ATL_6203. In other such embodiments, the antibody or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence VSYGGTENV (SEQ ID NO: 162). Some examples of antibodies according to these embodiments include ATL6378.

[0017] The VH domain can be a human VH domain. The antibody or fragment thereof can have the framework sequence of ATL_0006199VH:

[0018] EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCSP(SEQ ID NO:169)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101).

[0019] The antibody or fragment thereof may have the framework sequence of ATL_0006200 VH (as well as ATL6374 VH and ATL_6194 VH):

[0020] EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVP(SEQ ID NO:170)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101).

[0021] The antibody or fragment thereof may have the framework sequence of ATL_0006202 VH:

[0022] EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCSP(SEQ ID NO:171)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101).

[0023] The antibody or fragment thereof may have the framework sequence of ATL_0006203 VH (as well as ATL_5895VH and ATL_6204VH):

[0024] EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCIP(SEQ ID NO:172)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101).

[0025] The antibody or fragment thereof may have the framework sequence of ATL_0006205 VH (as well as ATL_6375VH, ATL_6376VH, ATL6377VH and ATL_6378VH):

[0026] EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCVP(SEQ ID NO:173)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101).

[0027] The antibody or fragment thereof may have the framework sequence of ATL_006195VH:

[0028] EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTP(SEQ ID NO:180)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101).

[0029] In some embodiments, the VL domain is a human VL domain. In some embodiments, the antibody or fragment thereof has the VL domain framework sequence of ATL_0005895 VL:

[0030] QSALTQPRSVSGSPGQSVTISC(SEQ ID NO:131)-[CDRL1]-WYQQHPGKAPKLMIY(SEQ ID NO:133)-[CDRL2]-GVPDRFSGSKSGATASLTISGLQAEDEADYYC(SEQ ID NO:138)-[CDRL3]-FGGTTKLTVL(SEQ ID NO:139).

[0031] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the VH domain are in germline framework. In some embodiments, the LCDR1, LCDR2, and LCDR3 of the VL domain are in germline framework.

[0032] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of the following:

[0033] ATL5895 VH (SEQ ID NO: 7), ATL_6204VH (SEQ ID NO: 7), ATL_6199 VH (SEQ ID NO: 144),

[0034] ATL6374 VH (SEQ ID NO: 148), ATL_6194 VH (SEQ ID NO: 145), ATL_6375VH (SEQ ID NO: 145),

[0035] ATL6200 VH (SEQ ID NO: 145), ATL_6202 VH (SEQ ID NO: 146), ATL_6203VH (SEQ ID NO: 147),

[0036] ATL6205 VH (SEQ ID NO: 148), ATL_6376 VH (SEQ ID NO: 175), ATL_6377 VH (SEQ ID NO: 176),

[0037] ATL6195VH (SEQ ID NO: 179), ATL_6378 VH (SEQ ID NO: 176).

[0038] In some such embodiments, the light chain variable domain comprises the amino acid sequence of any of the following:

[0039]

[0040] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of ATL_5895VH (SEQ ID NO: 7), ATL_6376VH (SEQ ID NO: 175), ATL_6377VH (SEQ ID NO: 176), or a sequence comprising at most 1, 2, or 3 mutations compared to any one of these sequences. In some such embodiments, the light chain variable domain comprises the amino acid sequence of any one of ATL_5895VL (SEQ ID NO: 8), ATL_6376VL (SEQ ID NO: 153), ATL_6377VL (SEQ ID NO: 154), or a sequence comprising at most 1, 2, or 3 mutations compared to any one of these sequences.

[0041] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0042]

[0043] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0044]

[0045] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0046]

[0047] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0048]

[0049] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0050]

[0051] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0052]

[0053] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0054]

[0055] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0056]

[0057] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0058]

[0059] In some such embodiments, the light chain variable domain sequence comprises the amino acid sequence

[0060]

[0061] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0062]

[0063] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0064]

[0065] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0066]

[0067] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0068]

[0069] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0070]

[0071] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0072]

[0073] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0074]

[0075] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0076]

[0077] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0078]

[0079] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0080]

[0081] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0082]

[0083] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0084]

[0085] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0086]

[0087] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0088]

[0089] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0090]

[0091] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0092]

[0093] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0094]

[0095] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0096]

[0097] In some embodiments, the heavy chain variable domain comprises a variable domain comprising an amino acid sequence that has at least 95% sequence identity, or comprises at most 1, 2, or 3 substitutions, compared to any one of the above heavy chain variable domains (SEQ ID No: 7, 148, 145, 175, 176, 144, 146, 147, 179), and / or the light chain variable domain comprises an amino acid sequence that has at least 90%, at least 95% sequence identity, or comprises at most 1, 2, 3, 4, or 5 substitutions, compared to any one of the above light chain variable domains (SEQ ID No: 8, 153, 154, 159, 155, 156).

[0098] In some embodiments, the HTT protein is human HTT or mouse HTT. In some embodiments, the isolated antibody or fragment thereof binds to a region located within exon 1 of HTT. In some embodiments, the isolated antibody or fragment thereof binds to HTT or a fragment thereof comprising at least a portion of exon 1, and the EC50 value is lower than that of a reference antibody, as measured by sandwich ELISA. In some embodiments, HTT has 25Q repeats or 48 repeats in exon 1. In some embodiments, the isolated antibody or fragment thereof has improved binding to mutated and / or aggregated HTT proteins compared to non-mutated and / or non-aggregated HTT proteins. In some embodiments, the relative binding to mutated and / or aggregated HTT and non-mutated and / or non-aggregated HTT is measured by determining the ratio of the EC50 value of the HTT protein or fragment thereof comprising 25Q repeats in exon 1 to the EC50 value of the HTT protein or fragment thereof comprising 48Q repeats in exon 1. In some embodiments, the HTT protein or fragment thereof is a fragment corresponding to exon 1. In some embodiments, the EC50 is as measured by sandwich ELISA. In some embodiments, the isolated antibody or fragment thereof has a higher relative binding to mutant and / or aggregated HTT and non-mutated and / or non-aggregated HTT compared to a reference antibody (e.g., such as ATL_0005059). In some embodiments, the ratio of the EC50 of the isolated antibody or fragment thereof binding to an HTT protein comprising a 25Q repeat in exon 1 or a fragment thereof and the EC50 of binding to an HTT protein comprising a 48Q repeat in exon 1 or a fragment thereof is at least 1.15, at least 1.18, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9 or at least 2, as measured by sandwich ELISA. The HTT or HTT fragment comprising a 48Q repeat may comprise the sequence of SEQ ID NO: 44 or 46. The HTT or HTT fragment comprising a 25Q repeat may comprise the sequence of SEQ ID NO: 43 or 45. Sandwich ELISA can be performed as described herein (Examples, Materials and Methods).Thus, the antibodies described herein may be able to preferentially bind to mutant HTT, thereby reducing the seeding propensity of mutant HTT (ie, reducing mutant HTT aggregation).

[0099] The isolated antibody or fragment thereof can increase the phagocytosis of cells to HTT protein or its fragment comprising exon 1 containing 48Q repeats. The cell can be a microglia, optionally a human microglia derived from iPSC. The isolated antibody or fragment thereof can increase the phagocytosis of cells to HTT protein or its fragment comprising exon 1 containing 48Q repeats in a dose-dependent manner. The increased phagocytosis can be measured by detecting the phagocytosis of beads coated with HTT protein or fragments coated with a pH-sensitive fluorescent dye. The increased phagocytosis can be measured as described herein (Materials and Methods).

[0100] The isolated antibody or fragment thereof can reduce the aggregation rate of HTT protein or its fragment comprising exon 1 containing 48Q repeats in a cell-free assay. The reduced aggregation rate can be measured using a FRASE assay. The reduced aggregation rate can be measured as described in (Materials and Methods) herein. Immunodepletion of a solution comprising HTT protein or its fragment with an isolated antibody or fragment thereof can result in a Δt50 of at most 0.1, at most 0.2, or at most 0.3 for aggregation of HTT protein or its fragment. Aggregation of HTT protein or its fragment is measured in the presence of HTT fibrils (e.g., from recombinant HTT) and / or brain homogenate from one or more R6 / 2 mice.

[0101] In some embodiments, the reference antibody comprises: (a) a heavy chain variable (VH) domain having the following CDRs: i. HCDR1 having the amino acid sequence NAWMN (SEQ ID NO: 35); ii. HCDR2 having the amino acid sequence HIRTQAEGGTSDYAAPVKG (SEQ ID NO: 36); iii. HCDR3 having the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3); and (b) a light chain variable (VL) domain having the following CDRs: i. LCDR1 having the amino acid sequence TGASSDVGTYDLVS (SEQ ID NO: 37); ii. LCDR2 having the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 having the amino acid sequence CSYAGYSTV (SEQ ID NO: 38). In some embodiments, the reference antibody is NI-302.8F1 described in US Pat. No. 11,401,325.

[0102] In some embodiments, the isolated antibody or fragment thereof binds to a mutant and / or aggregated HTT protein as determined by measuring immunoprecipitation of mutant HTT with the isolated antibody or fragment thereof. In some embodiments, the mutant HTT is an HTT protein or fragment thereof. In some embodiments, the mutant HTT is an HTT fragment comprising exon 1. In some embodiments, the mutant HTT is an HTT protein or fragment comprising more than 35 glutamine residues in its polyQ tract. Preferential binding to a more aggregation-prone, potentially more pathological form of HTT can advantageously inhibit the template potential of aggregated / mutant HTT, thereby inhibiting the progression of diseases associated with protein aggregation. For example, the isolated antibodies or fragments described herein can bind to a mutant and / or aggregated HTT protein (HTT) comprising 110 CAG repeats. 110 ). As another example, the mutant and / or aggregated HTT protein can immunoprecipitate a mutant and / or aggregated HTT protein comprising about 120 CAG repeats (e.g., extracted from a cell or tissue sample (e.g., a sample from an R6 / 2 transgenic mouse)). R6 / 2 transgenic mice express the 5' end of the human HTT gene, including exon 1 having about 120 CAG repeats, and exhibit a neurological phenotype similar to that characteristic of HD in humans. As another example, the isolated antibodies or fragments as described herein can immunoprecipitate a mutant and / or aggregated HTT protein comprising about 115 to 150 CAG repeats (e.g., extracted from a cell or tissue sample (e.g., a sample from an R6 / 1 transgenic mouse)). R6 / 1 transgenic mice ubiquitously express a transgene comprising the 5' end of the mutant human HTT gene, which includes approximately 1 kb of 5' UTR sequence, exon 1 (carrying an expanded CAG repeat with 115 to 150 CAG repeats), and the first 262 bp of intron 1. R6 / 1 mice exhibit a progressive neurological phenotype that mimics many features of Huntington's disease (Mangiarinie et al; Cell; 1996), including the accumulation of aggregates over time (Hansson et al; EJN; 2001).

[0103] In some embodiments, the isolated antibody or fragment thereof reduces the aggregation of mutant HTT, wherein the aggregation of mutant HTT is measured as the presence and / or concentration of HTT aggregates in the brain of a transgenic mouse model for Huntington's disease. The mouse model can be an R6 / 1 mouse. Treatment of the mouse with the isolated antibody or fragment thereof for 12 weeks or longer can result in a statistically significant reduction in the concentration of HTT aggregates in the striatum and / or cortex. In some embodiments, the antibody or fragment binds to mutant HTT in vivo. The mutant HTT can be an HTT protein or fragment thereof comprising more than 35 glutamine residues or 115 to 150 glutamine residues in its polyQ tract, optionally comprising a fragment of exon 1. In some embodiments, the isolated antibody or fragment thereof does not reduce the level of non-mutated and / or non-aggregated HTT in the brain of a transgenic mouse model for Huntington's disease treated with the isolated antibody or fragment thereof, wherein the level of non-mutated and / or non-aggregated HTT is measured as the concentration of soluble and / or non-mutated HTT in the mouse, optionally wherein the mouse is an R6 / 1 mouse.

[0104] In some embodiments, the antibody or fragment thereof retains a monomer percentage greater than 95% or greater than 97% after incubation at -80°C, 4°C, 21°C, 40°C for 4 weeks and / or after 10× freeze-thaw cycles. In some embodiments, the antibody or fragment thereof binds to an HTT protein comprising exon 1 of HTT and 48 glutamine residues in its polyQ tract as assessed by ELISA after incubation at -80°C, 4°C, 21°C, and 40°C for 4 weeks and / or 10× freeze-thaw cycles. In some such embodiments, the binding is not significantly different from the binding of the antibody to the HTT protein before incubation and / or 10× freeze-thaw cycles.

[0105] In some embodiments, the isolated antibody or fragment thereof binds to HTT or a fragment thereof comprising at least a portion of exon 1 with an EC50 value of at most 15 nM, at most 12 nM, at most 10 nM, or at most 5 nM, as measured using a sandwich ELISA. HTT can have a 25Q repeat or a 48 repeat in exon 1. HTT or an HTT fragment can comprise the sequence of SEQ ID NO: 43, 44, 45, or 46, and the sandwich ELISA can be performed as described herein (Examples, Materials and Methods).

[0106] In some embodiments, the isolated antibody or fragment thereof recognizes an epitope in a region corresponding to exon 1 of the HTT gene. In some embodiments, the isolated antibody or fragment thereof recognizes an epitope located in the polyP region of HTT. In some embodiments, the isolated antibody or fragment thereof recognizes an epitope comprising the amino acid sequence QQQQPPPPPPPPPPP (SEQ ID NO: 47) or PQPQPPPPPPPPPPP (SEQ ID NO: 48). In some embodiments, the isolated antibody or fragment thereof is capable of crossing the blood-brain barrier. In some embodiments, the isolated antibody or fragment thereof is a bispecific antibody that further comprises a region that binds to the transferrin receptor. The bispecific antibody comprises an isolated antibody or antibody fragment according to any of the foregoing embodiments, such as a single-chain variable fragment (scFv) according to the first aspect, and a binding portion (e.g., scFv, nanobody, or aptamer) that binds to a brain receptor (e.g., transferrin receptor).

[0107] In some embodiments, the isolated antibody or antibody fragment comprises a single-chain variable fragment (scFv) or a fragment antigen-binding region (fragment antigen-binding, Fab). In some embodiments, the isolated antibody or antibody fragment comprises an antibody constant region. In some embodiments, the isolated antibody comprises a whole antibody. Optionally, the whole antibody can be an IgG1 antibody.

[0108] In another aspect, the present disclosure provides an isolated antibody VH domain of the isolated antibody or antibody fragment according to the preceding aspect.

[0109] In another aspect, the disclosure provides an isolated antibody VL domain of the isolated antibody or antibody fragment according to the first or second aspect.

[0110] In another aspect, the present disclosure provides a composition comprising the isolated antibody, antibody fragment, antibody VH domain or antibody VL domain according to the first or second aspect.

[0111] In another aspect, the disclosure provides a host cell transformed in vitro with a nucleic acid molecule encoding the antibody or antibody fragment thereof according to the first or second aspect.

[0112] In another aspect, the present disclosure provides a method for producing an antibody or antibody fragment (including, for example, an antibody VH or VL domain) according to any embodiment of the first or second aspect, the method comprising culturing a host cell transformed in vitro with a nucleic acid molecule encoding the antibody or antibody fragment under conditions suitable for producing the antibody or antibody fragment. The method may further comprise isolating and / or purifying the antibody or antibody fragment. The method may further comprise formulating the antibody or antibody fragment into a composition comprising at least one additional component.

[0113] In another aspect, the disclosure provides a DNA molecule or set of DNA molecules encoding the antibody or antibody fragment thereof according to the first or second aspect.

[0114] In another aspect, the disclosure provides a vector or set of vectors encoding a DNA molecule according to the first or second aspect.

[0115] In another aspect, the present disclosure provides a host cell comprising a vector or set of vectors according to the first or second aspect.

[0116] In another aspect, the present disclosure provides a method for treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antibody fragment thereof according to the first or second aspect. The treatment can prevent and / or reduce seeding and / or aggregation of mutant HTT in the subject. The disease or condition can be Huntington's disease, Alzheimer's disease, or frontotemporal dementia. In some embodiments, the treatment comprises administering an additional therapeutic agent simultaneously or sequentially with the isolated antibody or antibody fragment. In some embodiments, the treatment prevents and / or reduces aggregation of mutant HTT in the subject (including, for example, in the brain of the subject) without reducing the level of non-mutated and / or non-aggregated HTT in the subject (including, for example, in the brain of the subject).

[0117] In another aspect, the present disclosure provides use of the isolated antibody or antibody fragment thereof according to the first or second aspect in the preparation of a medicament for treating a condition or disease, wherein the condition or disease may be Huntington's disease, Alzheimer's disease, or frontotemporal dementia.

[0118] In another aspect, the present disclosure provides a composition comprising an isolated antibody or antibody fragment thereof according to the first or second aspect. The composition may comprise a pharmaceutically acceptable excipient, carrier, or vehicle. The composition may be used to treat a disease or condition. The disease or condition may be Huntington's disease, Alzheimer's disease, or frontotemporal dementia.

[0119] In another aspect, the disclosure relates to a method of diagnosing or monitoring the progression of a disease or condition characterized by the presence of mutated and / or aggregated HTT protein in a patient, the method comprising exposing a sample obtained from the patient to an antibody or fragment thereof as described herein.

[0120] In another aspect, the present disclosure provides a method for determining the effect of a treatment (e.g., such as a drug) on aggregated HTT protein present in a patient, the method comprising exposing a sample obtained from the patient to an antibody or fragment thereof as described herein. The method of the foregoing aspect may comprise determining the level of HTT protein in a sample from the patient by detecting the antibody or fragment thereof, or the binding between the HTT protein and the antibody or fragment thereof. The method may comprise comparing the determined level with a predetermined threshold or a level determined for one or more control samples. The sample may be a blood sample or a cerebrospinal fluid sample.

[0121] In another aspect, the present disclosure provides a method of preventing or reducing seeding and / or aggregation of mutant HTT in a subject in need thereof, wherein the method comprises administering to the subject a therapeutic amount of an isolated antibody or antibody fragment thereof.

[0122] The present disclosure also expressly includes combinations of the described aspects and preferred features, except where such combinations are expressly not permitted or expressly avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Some embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0124] Figure 1 schematically illustrates the method for unbiased antibody discovery and target identification. A. Antibody identification workflow. B. Alignment of seven antibodies discovered in an Alzheimer's patient cohort that display CDR3 sequences homologous to a known HTT binder (NI-302.8F1 described in US Patient No. 11,401,325B2).

[0125] Figure 2 schematically illustrates the phage display selection process. A. Generation of functional scFv libraries. Suitable phagemids containing VL sub-libraries are designed and produced from a healthy donor group. VH derived from AD recovery individuals is cloned upstream of the VL sub-library to produce a functional (fused VH and VL) scFv library. B. Phages of the AD recovery library displaying scFV are used for selection. Two or three rounds of selection are performed on the fixed HTT exon 1 antigen (human HTT exon 1 with 48Q repeats, see Table 1 for sequence), wherein each round of selection narrows the scFv library to those with antigen binding properties. Phage ELISA is used to determine target antigen binding, and the bound unique scFv is converted into IgG1 for determining antigen binding.

[0126] Figure 3 shows ELISA results for antibodies of the present disclosure. A. Phage ELISA of binding of phage displaying antibody fragments corresponding to selected antibody sequences to human mutant HTT (mHTT) with 48Q repeats (in each subsection of the figure, different colored bars represent different concentrations of mHTT: from left to right: 10 μg / ml, 2 μg / ml, and 0.4 μg / ml). B. IgG1 antibody ELISA against human HTT exon 1 (48Q) protein, showing strong dose-dependent binding to 5 of the 7 IgG1 converted antibodies tested.

[0127] Figure 4 shows VH paired with phage-displayed VL. A. The VH sequence identified as convergent in AD recovery individuals is designated ATL_0005042 (ATL_5042). The homologous HTT binding antibody is CA_0000274 (also known as ATL_0005059 or NI-302.8F1). B. Following phage display selection and sequence analysis against HTT exon 1, the functionally paired VL (ATL_0005331-5335) was aligned with the CA_0000274 VL.

[0128] Figure 5 The representation of peptides in the peptide array used for HTT epitope mapping is shown. Human IgG1 antibodies ATL_0005331, ATL_0005335 and ATL_0005566 were immunized against sequences of human, cynomolgus monkey (cyno) and mouse huntingtin exon 1. Epitope mapping and peptide screening: The huntingtin protein sequence was converted into a 15-amino acid linear peptide, of which a 14-amino acid peptide-peptide overlap was used for high-resolution epitope data.

[0129] Figure 6Results of epitope mapping and peptide screening for ATL_5331 are shown. Upper panel: Intensity plot showing corrected intensity values for human, cynomolgus monkey_1, cynomolgus monkey_2 (possibly two different sequences of cynomolgus monkey huntingtin protein sequences), and mouse huntingtin protein sequences, sorted from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein. Fluorescence intensity values for IgG tested at 1 μg / ml are plotted in green, and those for IgG tested at 10 μg / ml are plotted in red, with 1000 increments for each intensity value to aid visualization. The X-axis represents the type of HTT. Antibody ATL_0005331 showed two different signals on HTT exon 1. Many antibody responses were observed against epitope-like sequence patterns formed by adjacent peptides with a common motif at each site. Lower panel: Epitope mapping for ATL_0005331 based on human, cynomolgus monkey_1, cynomolgus monkey_2, and mouse huntingtin protein sequences. The amino acids from the HTT exon 1 derived peptides to which ATL_0005331 showed binding are shown in bold, and the peptides showing the highest relative binding at each site of the different species of HTT exon 1 are underlined.

[0130] Figure 7 Results of epitope mapping and peptide screening for ATL_5335 are shown. Upper panel: Intensity plot showing corrected intensity values for human, cynomolgus monkey_1, cynomolgus monkey_2 (possibly two different sequences of cynomolgus monkey huntingtin protein sequences), and mouse huntingtin protein sequences, sorted from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein. Fluorescence intensity values for IgG tested at 1 μg / ml are plotted in green, and those for IgG tested at 10 μg / ml are plotted in red, with 2000 increments for each intensity value to aid visualization. The X-axis represents the species of HTT. Antibody ATL_0005331 showed two different signals on HTT exon 1. Many antibody responses were observed against epitope-like sequence patterns formed by adjacent peptides with a common motif at each site. Lower panel: Epitope mapping for ATL_0005335 based on human, cynomolgus monkey_1, cynomolgus monkey_2, and mouse huntingtin protein sequences. The amino acids from the HTT exon 1 derived peptides to which ATL_0005331 showed binding are shown in bold, and the peptides showing the highest relative binding at each site of the different species of HTT exon 1 are underlined.

[0131] Figure 8Results of epitope and peptide screening for ATL_5566 are shown. Upper panel: Intensity plot showing corrected intensity values for human, cynomolgus monkey_1, cynomolgus monkey_2 (possibly two different sequences of cynomolgus monkey huntingtin protein sequences), and mouse huntingtin protein sequences, sorted from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein. Fluorescence intensity values for IgG tested at 1 μg / ml are plotted in green, and those for IgG tested at 10 μg / ml are plotted in red, with 1000 added to each intensity value to aid visualization. The X-axis represents the type of HTT. Antibody ATL_0005566 showed two different signals on HTT exon 1. Many antibody responses were observed against epitope-like sequence patterns formed by adjacent peptides with a common motif at each site. Lower panel: Epitope mapping for ATL_0005566 based on human, cynomolgus monkey_1, cynomolgus monkey_2, and mouse huntingtin protein sequences. The amino acids from the HTT exon 1 derived peptides to which ATL_0005566 showed binding are shown in bold, and the peptides showing the highest relative binding at each site of the different species of HTT exon 1 are underlined.

[0132] Figure 9 The workflow of 3-week stability study, low pH maintenance and freeze-thaw cycle test is shown. The six specified antibodies are standardized to 5 mg / ml and subsequently used for 3-week stability study, freeze-thaw study or low pH maintenance study. Quality control analysis is carried out, including SEC-HPLC (size exclusion high performance liquid chromatography), SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), cIEF (capillary isoelectric focusing) and thermal shift analysis.

[0133] Figure 10 shows the results of a 3-week stability study or freeze-thaw cycle: SEC-HPLC. SEC-HPLC chromatograms of the six designated antibodies (A: ATL_5331, ATL_5334, ATL_5335; B: ATL_5555, ATL_5556, ATL5557) after incubation at designated temperatures (-80°C, +4°C, +21°C, +40°C) for three weeks or five freeze-thaw cycles.

[0134] Figure 11 Results of a low pH retention study are shown: SEC-HPLC. The antibody was prepared at a concentration of 5 mg / ml in PBS. Acetic acid was added dropwise to pH 3.5 and Tris base (pH 11) was added to neutralize the solution to pH 7.2 to 7.4. Chromatograms of ATL_5331 and ATL_5335 at 0, 15, 30, 60, or 120 minutes after pH neutralization.

[0135] Figure 12 shows the results of charge heterogeneity assessment by cIEF. A. pI (isoelectric point) of each of the six antibodies before the 3-week stability study. B-1 to B-3. pI of each of the six antibodies after the 3-week stability study at the indicated temperature. C1 to C3. pI of each of the six antibodies after the 3-week stability study at -80°C or 5 freeze-thaw cycles (5×FT).

[0136] Figure 13 Results of HTT pharmacokinetic (PK) studies are shown. ATL_0005335 serum levels in mice measured by ELISA at 0, 1, 4, 8, 24, 72 and 144 hours after treatment with 10 or 20 mg / Kg.

[0137] Figure 14 The workflow for antibody variant panel classification to identify lead antibodies is schematically shown.

[0138] Figure 15 Results of analysis of recombinant HTT exon 1 ELISA (48Q GST - i.e., HTT exon 1 48Q with a glutathione S-transferase tag for purification of the antigen by affinity chromatography) (bars) and thermal stability of variant antibodies (dots) are shown. Melting temperatures were measured using SYPRO Orange.

[0139] Figure 16 shows the results of HTT sandwich ELISA for ATL_5895, ATL_5901, ATL5567, and ATL_5059 (as shown in the top row of the table in each figure). A to C. HTT exon 1 25Q ELISA replicate 1. D to G. HTT exon 1 25Q ELISA replicate 2. H to J. HTT exon 1 25Q ELISA replicate 3. K to N. HTT exon 1 48Q ELISA replicate 1. O to R. HTT exon 1 48Q ELISA replicate 2. S to V. HTT exon 1 48Q ELISA replicate 3.

[0140] Figure 17 shows the results of HTT immunoprecipitation. A. In the U-2OS cell line expressing 110 CAG repeats resulting in a high molecular weight HTT species (denoted as "HTT 110 ”) and the parental (control) cell line (denoted as “HTT WTA. ATL_5335 was tested for immunoprecipitation of HTT using homogenate from R6 / 2 mouse brain tissue (denoted as "R62 brain homogenate") or homogenate from non-transgenic brain tissue (denoted as "non-transgenic brain homogenate") to test immunoprecipitation of HTT using ATL_0005895, ATL_0005901, and ATL_0005567. Anti-HTT MW8 antibodies (MABN2529Sigma) and 1C2 (MAB1574Sigma-Aldrich) were used for detection. C. ATL_0005895 was tested for immunoprecipitation of HTT using homogenate from human Huntington's disease brain tissue. Anti-HTT antibodies HD1 and MW1 (MABN2427Millipore) were used for detection. D. The immunoprecipitation of HTT using Image J analysis software (NIH) was performed. Figure 17C Densitometric analysis of blots was performed.

[0141] Figure 18 shows the results of a FRET-based mHTT (FRASE) assay to assess the ability of antibodies to bind to seed-competent HTT (48Q) species and affect HTT aggregation. (A) shows the aggregation rate (Δt50 value) of seed-competent HTT (mHTT) in the presence of fibrils produced from recombinant HTT after immunodepletion of seeds using specified antibodies (ATL5895; ATL5901; MW8 and MW1). The data shown are representative examples from three biological replicates. Error bars represent + / -SD, from n=3 technical replicates in a single biological replicate. (B) shows the aggregation rate (Δt50 value) of a certain amount of R6 / 2 brain (referred to as "seed" in the figure) added as seed-competent HTT in the presence of brain homogenate of R6 / 2 mouse brain after immunodepletion of seeds using specified antibodies. Seeds were immunodepleted using the indicated antibodies (ATL5895; ATL5901; MW8 and MW1) on protein G beads (R6 / 2 "seeds" were incubated with antibodies on protein G beads for 1 hour at 4 degrees, followed by removal of the antibody-bound beads and seeds (if the antibody had bound to the seeds), and the remaining solution was used for the FRASe aggregation assay. The data shown represent 3 biological replicates, each using brains from a different R6 / 2 mouse. Error bars represent + / - SD, n = 3 technical replicates in a single biological replicate. These data demonstrate that immunodepletion of seeds with antibodies ATL_0005895 and ATL_0005901 reduced the ability of HTT seeds from both sources (fibrils from recombinant HT and brain homogenates from R6 / 2 mice) to increase the rate of aggregation in vitro. MW8 (Millipore; MABN2529) binds to aggregated HTT and also achieves this effect to some extent. MW1 (Millipore; MABN2427) binds to the PolyQ region of HTT and is unable to achieve this effect.

[0142] Figure 19 Figure 5 shows a dose-dependent increase in phagocytosis of 48QHTT-coated beads by iPSC-derived microglia in the presence of ATL5895. Phagocytosis of Q48HTT exon 1-coated beads by induced pluripotent stem cell (iPSC)-derived microglia was measured in the presence of ATL5895 or fluorescein-conjugated human IgG1 isotype control antibody ATL5338. TMUptake of red beads. ATL_5895 increased the total red area signal over time compared to the isotype control antibody. The figure shows the area under the curve (AUC) calculated from the total red fluorescence signal per well during the 4-hour reaction, relative to the antibody concentration in log nM.

[0143] Figure 20 shows the results of in vivo pharmacokinetic (PK) studies of antibodies of the present disclosure. (A) shows ATL_0005567 and 5901 serum levels in mice detected by ELISA at 0, 1, 4, 8, 24, 72, and 144 hours after treatment with 10 mg / Kg antibody by intraperitoneal (IP) injection. (B) shows ATL_0005895 serum levels in mice detected by ELISA at 0, 1, 4, 8, 24, 72, and 144 hours after treatment with 1, 10, or 60 mg / Kg antibody by IP injection. (C) shows cerebrospinal fluid (CSF) levels in mice detected by ELISA at 4 and 144 hours after treatment with 1, 10, and 60 mg / kg antibody by IP injection. ATL_0005895 1 mg / kg and 10 mg / Kg CSF levels at 144 hours were below the limit of quantitation of the assay.

[0144] Figure 21 shows the results of an indirect ELISA of the indicated antibodies binding to HTT exon 1 48Q or a lysozyme control. (A) ATL_6199, (B) ATL_6200, (C) ATL_6202, (D) ATL_6203, (E) ATL_6204, (F) ATL_6205, (G) ATL_6194, (H) ATL_6195. Absorbance was measured at 450 nm.

[0145] Figure 22 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Replicate 1 was performed using an antibody starting concentration of 133 nM, and the results are shown in (A) to (F), (M) to (N). (A) ATL_6199, (B) ATL_6200, (C) ATL_6202, (D) ATL_6203, (E) ATL_6204, (F) ATL_6205, (M) ATL_6194, (N) ATL_6195. Replicate 2 was performed using an antibody concentration of 400 nM, and the results are shown in (G) to (L). (G) ATL_6199, (H) ATL_6200, (I) ATL_6202, (J) ATL_6203, (K) ATL_6204, (L) ATL_6205, (O) ATL_6194, (P) ATL_6195. Absorbance was measured at 450 nm.

[0146] Figure 23 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. (A) ATL_6183, (B) ATL_6184, (C) ATL_6185, (D) ATL_6186, and (E) control antibody ATL_5338. Absorbance was measured at 450 nm.

[0147] Figure 24 shows the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. (A) Sequences of HFW1, HCDR1, HFW2, HCDR2, HFW3, HCDR3, and HFW4 of the specified antibodies as defined by Kabat. (B) Sequences of LFW1, LCDR1, LFW2, LCDR2, LFW3, LCDR3, and LFW4 of the specified antibodies as defined by Kabat. A-1 and A-2 show the heavy chain sequences of the antibodies described in Examples 1 to 10, 13 to 16, and 18. A-3 and A-4 show the heavy chain sequences of the antibodies described in Examples 11 to 12. A-5 shows the heavy chain sequences of the antibodies described in Examples 17 to 18. B-1 and B-2 show the light chain sequences of the antibodies described in Examples 1 to 10, 13 to 16, and 18. B-3 and B-4 show the light chain sequences of the antibodies described in Examples 11 to 12. B-5 shows the light chain sequences of the antibodies described in Examples 17 to 18.

[0148] Figure 25 shows the results of live animal PET / CT scans using radiolabeled antibodies and the results of gamma counting assays. (A and B) Percent injected dose (%ID) per gram of blood as measured by gamma counting at 0 to 168 hours after dosing in C57BL / 6J and R6 / 1 mice in the (A) 11- to 12-week-old group and (B) 14- to 15-week-old group. (C and D) 168 hours after dosing in C57BL / 6J (left) and R6 / 1 (right) mice in (C) 11- to 12-week-old and (D) 14- to 15-week-old groups. 89 Ex vivo biodistribution of Zr-Df-ATL5895, as assessed by gamma counting analysis. (E and F) Brain biodistribution of Zr-Df-ATL5895 over 168 hours in 14- to 15-week-old cohorts of C57BL / 6J and R6 / 1 mice, as determined by PET / CT imaging. (E) Representative coronal, sagittal, and transverse PET / CT images of WT and R6-1 mice.

[0149] Figure 26 shows the results of immunoassays (meso scale discovery (MSD) assays) evaluating the effect of ATL_5895 (ATLX_1095) on HTT aggregate load in the striatum and cortex of R6 / 1 mice. (A) Aggregated HTT in the striatum and cortex increases over time. (B) Twelve weeks of ATL_5895 (ATLX-1095) treatment of R6 / 1 mice results in a significant decrease in HTT aggregates (MW8 / 4C9+) in the striatum and cortex. (C) ATL_5895 (ATLX-1095) treatment does not affect the levels of mutant soluble HTT over time. (D) ATL_5895 (ATLX-1095) does not affect the levels of endogenous mouse HTT over time.

[0150] Figure 27 shows the results of the manufacturability study of ATL_5895 (ATLX_1095). A. SEC-HPLC chromatogram of a 4-week thermal stability study sample of ATL_5895-002. The sample was run on a Zorbax GF-250 SEC-HPLC column (Agilent). B. SEC-HPLC chromatogram of a 10× freeze-thaw sample of ATL_5895-002. The sample was run on a TSK gel G3000 SWxl column (TOSOHBioscience). C. cIEF electropherogram of a 4-week thermal stability study sample of ATL_5895-002. D. cIEF electropherogram of a 10× freeze-thaw sample of ATL_5895-002 and an unstressed control sample. E. Reduced CE-SDS profile of the thermal stability study sample of ATL_5895-002 and a 10× freeze-thaw cycle. F. HTT exon-1 sandwich ELISA of ATL_5895-002 4-week thermal stability study samples and 10× freeze-thaw cycle samples. G. Melting temperature (Tm1 / Tm2) and aggregation temperature (Tagg) of 5 mg / mL ATL_0005895-002 measured in 20 mM histidine-acetate, 150 mM NaCl (pH 5.5). H. SEC-HPLC chromatograms of ATL_5895 initial (T0) solubility study samples at 11.90 mg / mL, 23.91 mg / mL, 44.95 mg / mL, and 89.96 mg / mL. I. SEC-HPLC chromatograms of ATL_5895 solubility study samples at 11.90 mg / mL, 23.91 mg / mL, 44.95 mg / mL, and 89.96 mg / mL after incubation at 21°C for 1 week.

[0151] Figure 28 Figure 2 shows the results of direct ELISA studies of binding of ATL5895, ATL6376, and ATL6377 to mouse HTT protein. Human lysozyme was used as a control antigen. ATL5338 was used as a negative isotype control.

[0152] Figure 29 shows the results of a study of affinity-optimized antibodies binding to HTT exon 1 or mutant HTT exon 1 48Q. A. HTT exon 1 epitope peptide binding responses for the indicated mAbs measured by Octet, with a concentration of 15 nM for each mAb tested. The binding responses confirmed that the mAbs bound to HTT exon 1 and showed an increased binding propensity compared to the parent ATL_5895. B. HTT exon 1 epitope peptide binding responses for the indicated mAbs measured by Octet, with a concentration of 15 nM for each mAb tested. The binding responses confirmed that the mAbs bound to HTT exon 1 and showed an increased binding propensity compared to the parent ATL_5895. C. Mutant HTT exon 1 binding responses for the indicated mAbs measured by Octet, with a concentration of 25 nM for each mAb tested. The binding response confirmed that the mAb bound to HTT exon 1 and showed an increased binding propensity compared to the parental ATL_5895. DETAILED DESCRIPTION

[0153] Some aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0154] Disclosed herein are antibodies and fragments thereof that specifically bind to a huntingtin (HTT) protein or fragments thereof. This disclosure relates to antibodies described herein that are designated by reference numbers as "ATL_000xxxx," "ATL_xxxx," or "xxxx," where "xxxx" is a four-digit reference number specific to the antibody described herein. All of the above symbols are used interchangeably to refer to the same antibody or portion thereof (e.g., a VH, VL, or portion thereof of an antibody). For example, antibody ATL_0005895 is referred to herein interchangeably as ATL_5895 and 5895.

[0155] As used herein, an antibody that is capable of "specifically binding" or "binding specifically to" a target is an antibody that is capable of binding through association of an epitope within the target through an epitope recognition site. This is distinguished from non-specific binding, such as Fc-mediated binding, ionic and / or hydrophobic interactions. In other words, an antibody that specifically binds to a target recognizes and binds to a specific protein structure within the target, rather than proteins in general.

[0156] And it is widely distributed throughout the CNS.

[0157] Huntington's disease (HD) is a soluble 3144 amino acid (384 kDa) protein (in its non-expanded form) that is widely distributed throughout the central nervous system (CNS) and is associated with the neurodegenerative disorder Huntington's disease (HD). HD is caused by the expansion of the trinucleotide repeat CAG in exon 1 of HTT, which encodes a polyglutamine (polyQ) tract near the N-terminus of the toxic mutant huntingtin protein (mHTT). In healthy individuals, the length of the CAG repeat is typically 9 to 35 CAG repeats, while repeat numbers exceeding 40 lead to disease expression. CAG repeats of 36 to 39 are associated with reduced penetrance, whereby some individuals develop HD while others do not.

[0158] The elongated polyQ bundles in mutant huntingtin proteins cause protein misfolding and lead to reduced solubility. These insoluble aggregates of mHTT are highly toxic to neurons, ultimately leading to neuronal cell death. Toxic accumulation of mHTT occurs in different parts of the brain, and aggregation can occur in the nucleus, cytoplasm, and extracellularly. In addition, mutant aggregates show "seeding" potential, that is, these proteins spontaneously aggregate, and when evaluated, for example, in cell-free assays, the aggregates accelerate the aggregation rate of mHTT, leading to the spread of pathological HTT in the CNS. Advantageously, the antibodies of the present disclosure can bind to toxic extracellular mHTT and prevent the spread of mHTT by removing aggregated mHTT and / or inhibiting the seeding potential of mHTT. The human gene encoding HTT (Gene ID: 3064) is located at 4p16.3 and is large, spanning 180kb and consisting of 67 exons. Reference non-human HTT amino acid and coding sequences are available in public databases.

[0159] Reference human HTT exon 1 amino acid sequence is provided below as "wild-type huntingtin protein (HTT) exon 1" or "mutant huntingtin protein (HTT) exon 1" (see below and Table 1). However, as used herein, the terms "HTT" and "HTT exon 1" encompass truncations, derivatives, and variants of the HTT exon 1 sequences provided herein, and may refer to any protein having at least 80%, at least 90%, or at least 95% sequence identity with "wild-type huntingtin protein (HTT) exon 1" or "mutant huntingtin protein (HTT) exon 1" below. In some embodiments, the antibodies described herein are capable of specifically binding to a peptide or protein having or comprising the amino acid sequence of "wild-type huntingtin protein (HTT) exon 1" or "mutant huntingtin protein (HTT) exon 1" (including a full-length HTT protein containing the sequence, or a fragment of the protein containing the sequence), or a fragment thereof.

[0160] In some embodiments, the antibodies described herein are capable of specifically binding to an HTT protein or protein fragment comprising or consisting of an HTT variant amino acid sequence. In some embodiments, the HTT variant protein or fragment comprises an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95% or at least 99% identical to "wild-type huntingtin protein (HTT) exon 1" or "mutant huntingtin protein (HTT) exon 1". In some embodiments, the antibodies disclosed herein are capable of specifically binding to an HTT fragment comprising at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of an HTT amino acid sequence or an HTT variant sequence.

[0161] In some embodiments, the antibodies and fragments of the present disclosure bind to a region in exon 1 of HTT. Exon 1 of HTT may contain elongated polyQ repeats caused by expanded CAG repeats (e.g., more than 35 to 40 CAG repeats). Any HTT protein or fragment thereof containing a polyQ tract with 36 or more glutamines can be considered pathological, meaning that HD disease expression can occur. Therefore, as used herein, the term "mutant HTT" (mHTT) refers to an HTT protein or fragment thereof having at least 36 glutamines in its polyQ repeat region. Exon 1 HTT with 35 or fewer glutamines can be considered non-pathological. HTT proteins or fragments thereof containing 35 or fewer glutamines in the polyQ repeat region will be referred to as wild-type (WT) HTT. The terms "mutant HTT" and "aggregated HTT" are used interchangeably herein to refer to HTT with expanded polyQ tracts (also known as "high molecular weight HTT"), such as HTT with 36 or more glutamines.

[0162] In some embodiments, HTT exon 1 has a polyQ tract containing 9 to 35 glutamines. In some embodiments, the polyQ tract contains 25 glutamines. For example, human HTT exon 1 can have a polyQ tract containing 25 glutamines. Human HTT exon 1 having a polyQ tract containing 25 glutamines can have the following sequence (referred to as "wild-type huntingtin (HTT) exon 1"):

[0163] >human_HTT_exon_1_25Q (SEQ ID NO: 43)

[0164]

[0165] In some embodiments, HTT exon 1 has a polyQ tract containing more than 40 glutamines. In some embodiments, the polyQ tract contains 48 glutamines. For example, human HTT exon 1 can have a polyQ tract containing 48 glutamines. Human HTT exon 1 having a polyQ tract containing 48 glutamines can have the following sequence (referred to as "mutant huntingtin (HTT) exon 1"):

[0166] >human_HTT_exon_1_48Q (SEQ ID NO: 44)

[0167]

[0168] The binding region of the antibodies described herein may be located in the polyP region, polyQ / polyP region, P-rich region, C-terminal region and / or N-terminal region of HTT. In some embodiments, the antibody binds to an epitope within the polyP and / or polyQ / polyP tract of HTT. In some embodiments, the epitope comprises or is comprised of the amino acid sequence QQQQPPPPPPPPPPP (SEQ ID NO: 47) or PQPQPPPPPPPPPPPP (SEQ ID NO: 48) of human HTT, or a corresponding region in a homologous protein. The binding regions of the exemplary antibodies of the present disclosure to various HTT proteins are Figures 6 to 8 In some embodiments, the binding region of the antibodies described herein is located at Figure 6 、 7 Or any region in bold on 8. The region in HTT exon 1 is discussed by Angelopoulou, E., et al. (Exploring the role of high-mobility group box 1 (HMGB1) protein in the pathogenesis of Huntington's disease. J Mol Med 98, 325-334 (2020)). In some embodiments, the antibody binds to an epitope within HTT exon 1. In some embodiments, the antibody binds to an epitope within HTT exon 1 that does not contain a polyQ tract. In some embodiments, the antibody may not bind to proteins derived from genes other than HTT that contain CAG repeats. In other words, the antibodies of the present disclosure may advantageously not show off-target binding to other proteins expressed by CAG repeat genes, such as, for example, ataxins. In some embodiments, the antibodies of the present disclosure may bind to soluble forms of high molecular weight / aggregated HTT and HTT / wild-type HTT.

[0169] HTT may be human HTT or mouse HTT. Suitably, HTT may be human HTT. Unless the context indicates otherwise, HTT may refer to human HTT. In other embodiments, for example, when the individual to be treated is a non-human mammal, HTT may be non-human HTT.

[0170] The antibody or fragment thereof can bind to human HTT and can also bind to mouse (murine) HTT antigen. For example, the antibody or fragment thereof can also bind to the mouse HTT antigen having the amino acid sequence set forth in SEQ ID NO: 177. Cross-reactivity with the mouse HTT antigen can be determined by direct ELISA, such as that performed as described herein (Example 18, Materials and Methods).

[0171] The present disclosure relates primarily to antibody molecules, whether intact antibodies (e.g., IgG, e.g., IgG4) or antibody fragments (e.g., scFv, Fab, (single domain) dAbs). Antibody antigen-binding regions (also referred to as "antigen-binding portions") are provided, as are antibody VH and VL domains. Within the VH and VL domains, complementary determining regions (CDRs) are provided, which may be provided within different framework regions (FRs) to form VH or VL domains, depending on the circumstances. The antigen-binding site may be composed of an antibody VH domain and / or VL domain.

[0172] Antibodies according to the present disclosure may be provided in isolated form.The term "antibody" encompasses fragments or derivatives thereof, or synthetic antibodies or synthetic antibody fragments.

[0173] The antigen binding portion may be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment [ScFv]). Suitable monoclonal antibodies against the selected antigen can be prepared by known techniques, such as those disclosed in "Monoclonal Antibodies: A manual of techniques", H. Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications", J. G. R. Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 25 792-799).

[0174] The antibody or fragment thereof may be a monoclonal antibody. A monoclonal antibody (mAb) is a homogeneous antibody population that specifically targets a single epitope on an antigen.

[0175] Antibody fragments, such as Fab and Fab2 fragments, are also provided, as are genetically engineered antibodies and antibody fragments. The variable heavy (VH) and variable light (VL) domains of antibodies are involved in antigen recognition, a fact first confirmed by early protease digestion experiments. Further confirmation has been obtained by the "humanization" of rodent antibodies. Rodent-derived variable domains can be fused with human-derived constant domains so that the resulting antibody retains the antigenic specificity of the rodent parent antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

[0176] Antigen specificity is conferred by the variable domains and is independent of the constant domains, as known from experiments involving bacterial expression of antibody fragments, all of which contain one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules in which the VH and VL partner domains are linked by a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sd. USA 85, 5879); and single-domain antibodies (dAbs) containing a single V domain (Ward et al (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is found in Winter & Milstein (1991) Nature 349, 293-299.

[0177] The term "ScFv molecule" refers to a molecule in which the VH and VL partner domains are covalently linked, for example, by a flexible oligopeptide. Fab, Fv, ScFv, and dAb antibody fragments can all be expressed in and secreted from E. coli, making it easy to produce large quantities of the fragments. Whole antibodies and F(ab')2 fragments are "bivalent." The term "bivalent" means that the antibody and F(ab')2 fragment have two antigen-binding sites. In contrast, Fab, Fv, scFv, and dAb fragments are monovalent and have only one antigen-binding site.

[0178] Antibodies according to the present disclosure may be detectably labeled or at least capable of detection. For example, antibodies may be labeled with radioactive atoms or colored molecules or fluorescent molecules or molecules that can be easily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates, and radioactive labels. The binding moiety (antibody or fragment thereof) may be directly labeled with a detectable label, or it may be indirectly labeled. For example, the binding moiety may be an unlabeled antibody that can be detected by another self-labeled antibody. Alternatively, the second antibody may be combined with biotin, and the combination of labeled streptavidin and biotin is used to indirectly label the first antibody.

[0179] A "fragment" of an antibody may comprise any number of residues from the "parent" antibody while retaining target binding ability. A fragment may lack effector function, for example, relative to the parent, the fragment may be completely unable to bind to an Fc receptor or may exhibit reduced binding to an Fc receptor. A fragment is typically smaller than the parent antibody. A fragment may comprise 50%, 60%, 70%, 80%, 90%, 95% or more of the continuous or non-continuous amino acids of the parent antibody. A fragment may comprise 50, 100, 150, 200, 250, 300 or more of the continuous or non-continuous amino acids of the parent antibody. A fragment may comprise a deletion in the Fc region or a deletion in the Fc region. A fragment may retain the CDRs and / or variable domains of the parent antibody without alteration. In some embodiments, the fragment is a Fab fragment or a F(ab')2 fragment.

[0180] CDR sequences are described herein using the Kabat definition (Kabat, EA et al., Sequences of Proteins of Immunological Interest.).

[0181] Antibodies according to the present disclosure may have the CDRs of antibody ATL_5895, wherein:

[0182] i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1);

[0183] ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2);

[0184] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0185] iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4);

[0186] v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5);

[0187] vi. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6).

[0188] Antibodies according to the present disclosure may have a V of antibody ATL_5895. H and / or V L Sequence, where: (a) V of ATL_5895 H With the following sequence:

[0189]

[0190] and (b) V of ATL_5895 L With the following sequence:

[0191]

[0192] Antibodies according to the present disclosure may have the CDRs of antibody ATL_5901, wherein:

[0193] i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1);

[0194] ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2);

[0195] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0196] iv. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9);

[0197] v. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10);

[0198] vi. LCDR3 has the amino acid sequence SSYAGSSVV (SEQ ID NO: 11).

[0199] Antibodies according to the present disclosure may have a V of antibody ATL_5901. H and / or V L Sequence, where: (a) V of ATL_5901 H With the following sequence:

[0200]

[0201] and (b) V of ATL_5901 L With the following sequence:

[0202]

[0203] Antibodies according to the present disclosure may have the CDRs of antibody ATL_5567, wherein:

[0204] i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14);

[0205] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15);

[0206] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0207] iv. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16);

[0208] v. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17);

[0209] vi. LCDR3 has the amino acid sequence SSYAGFSTLV (SEQ ID NO: 18).

[0210] Antibodies according to the present disclosure may have a V of antibody ATL_5567. H and / or V L Sequence, where: (a) V of ATL_5567 H With the following sequence: (a)

[0211]

[0212] and (b) ATL 5567's V L With the following sequence:

[0213]

[0214] The antibodies of the present disclosure may have the CDRs of antibody ATL_5331, wherein:

[0215] i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14);

[0216] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15);

[0217] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0218] iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4);

[0219] v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5);

[0220] vi. LCDR3 has the amino acid sequence GSYAGTNNV (SEQ ID NO: 21).

[0221] Antibodies according to the present disclosure may have a V of antibody ATL_5331. H and / or V L Sequences, wherein: (a) VH of ATL_5331 (AC_0737) has the following sequence:

[0222]

[0223] and (b) ATL 5331's V L With the following sequence:

[0224]

[0225] Antibodies of the present disclosure may have the CDRs of ATL_5334:

[0226] i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14);

[0227] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15);

[0228] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0229] iv. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9);

[0230] v. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10);

[0231] vi. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23).

[0232] Antibodies according to the present disclosure may have a V of antibody ATL_5334. H and / or V L Sequences, wherein: (a) VH of ATL_5334 (AC_0737) has the following sequence:

[0233]

[0234] and (b) ATL 5334's V L With the following sequence:

[0235]

[0236] Antibodies of the present disclosure may have the CDRs of ATL_5335:

[0237] i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14);

[0238] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15);

[0239] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0240] iv. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16);

[0241] v. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17);

[0242] vi. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25).

[0243] Antibodies according to the present disclosure may have a V of antibody ATL_5335. H and / or V L Sequences, wherein: (a) VH of ATL_5335 (AC_0737) has the following sequence:

[0244]

[0245] and (b) V of ATL_5335 L With the following sequence:

[0246]

[0247] The antibodies of the present disclosure may have the CDRs of ATL_5555, wherein:

[0248] i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1);

[0249] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27);

[0250] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0251] iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4);

[0252] v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5);

[0253] vi. LCDR3 has the amino acid sequence GSYAGTNNV (SEQ ID NO: 21).

[0254] Antibodies according to the present disclosure may have a V of antibody ATL_5555. H and / or V L Sequences, wherein: (a) VH (AC_1269) of ATL_5555 has the following sequence:

[0255]

[0256] and (b) ATL_5555's V L With the following sequence:

[0257]

[0258] The antibodies of the present disclosure may have the CDRs of ATL_5556, wherein:

[0259] i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1);

[0260] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27);

[0261] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0262] iv. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9);

[0263] v. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10);

[0264] vi. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23).

[0265] Antibodies according to the present disclosure may have a V of antibody ATL_5556. H and / or V L Sequences, wherein: (a) VH of ATL_5556 (AC_1269) has the following sequence:

[0266]

[0267] and (b) V of ATL_5556 L With the following sequence:

[0268]

[0269] The antibodies of the present disclosure may have the CDRs of ATL_5557, wherein:

[0270] i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1);

[0271] ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27);

[0272] iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3);

[0273] iv. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16);

[0274] v. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17);

[0275] vi. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25).

[0276] Antibodies according to the present disclosure may have V H and / or V L Sequences, wherein: (a) VH of ATL_5557 (AC_1269) has the following sequence:

[0277]

[0278] and (b) V of ATL_5557 L With the following sequence:

[0279]

[0280] The antibodies of the present disclosure may have CDRs of any of the following:

[0281] ATL_6199, ATL_6200, ATL_6202, ATL_6203, ATL_6204, ATL_6205, ATL_6194, ATL_6195, ATL_6374, ATL_6375, ATL_6376, ATL_6377, ATL_6378, as provided in Table 1. Antibodies according to the present disclosure may have a V of any of the following: H and / or V L Sequence: Antibody

[0282] ATL_6199, ATL_6200, ATL_6202, ATL_6203, ATL_6204, ATL_6205, ATL_6194, ATL_6195, ATL_6374, ATL_6375, ATL_6376, ATL_6377, ATL_6378,

[0283] As provided below:

[0284] -ATL_6199 VH:

[0285]

[0286] and VL of ATL_6199:

[0287]

[0288] -VH of ATL_6200:

[0289]

[0290] VL of ATL_6200:

[0291]

[0292] -VH of ATL_6202:

[0293]

[0294] VL of ATL_6202:

[0295]

[0296] -VH of ATL_6203:

[0297]

[0298] VL of ATL_6203:

[0299]

[0300] -VH of ATL_6204:

[0301]

[0302] VL of ATL_6204:

[0303]

[0304] -VH of ATL_6205:

[0305]

[0306] VL of ATL_6205:

[0307]

[0308] -VH of ATL_6194:

[0309]

[0310] VL of ATL_6194:

[0311]

[0312] -VH of ATL_6195:

[0313]

[0314] VL of ATL_6195:

[0315]

[0316] - VH of ATL_6374:

[0317]

[0318] VL of ATL_6374:

[0319]

[0320] -VH of ATL_6375:

[0321]

[0322] VL of ATL_6375:

[0323]

[0324] -VH of ATL_6376:

[0325]

[0326] VL of ATL_6376:

[0327]

[0328] - VH of ATL_6377:

[0329]

[0330] VL of ATL_6377:

[0331]

[0332] - VH of ATL_6378:

[0333]

[0334] VL of ATL_6378:

[0335]

[0336] The antibodies of the present disclosure may have the CDRs of any one of antibodies ATL_6183, ATL_6184, ATL_6185, ATL_6186, as provided in Table 1. The antibodies according to the present disclosure may have the V H and / or V L Sequence, as provided below:

[0337] - VH of ATL6183:

[0338]

[0339] VL of ATL6183:

[0340]

[0341] - VH of ATL6184:

[0342]

[0343] VL of ATL6184:

[0344]

[0345] - VH of ATL6185:

[0346]

[0347] VL of ATL6185:

[0348]

[0349] - VH of ATL6186:

[0350]

[0351] VL of ATL6186:

[0352]

[0353] In an antibody according to the present disclosure, at least one of sequences (i) to (vi) may vary. A variant may have one, two, three, four, five or more (e.g., such as up to 10) amino acid substitutions in one or more of sequences (i) to (vi). In some embodiments, an antibody according to the present disclosure comprises a CDR having a sequence with 1, 2, or 3 substitutions compared to sequences (i) to (vi) of any of the above antibodies. For example, an antibody according to the present disclosure may comprise a CDR having the sequence of any of the above antibodies, except that 1, 2, or 3 of the CDRs comprise substitutions, wherein the total number of substitutions in all CDRs does not exceed 3.

[0354] The VH and VL chain CDRs 1 to 3 of any of the antibodies described above are also particularly useful for binding to a number of different framework regions. Thus, the light and / or heavy chains having CDRs 1 to 3 as described above may have alternative framework regions. Suitable framework regions are known in the art and are described, for example, in M. Lefranc & G. Le Franc (2001) "The Immunoglobulin Facts Book", Academic Press.

[0355] In the present specification, the antibody may have a VH and / or VL region comprising an amino acid sequence having a high percentage of sequence identity with the above-mentioned VH and / or VL amino acid sequence.

[0356] For example, antibodies according to the present invention include antibodies that bind to HTT and have a VH region comprising an amino acid sequence that has at least 70%, more preferably at least one of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the VH region of any of the above-mentioned antibodies (e.g., ATL5895, ATL5901 or 5567).

[0357] Alternatively or in addition, an antibody of the present disclosure may have a VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the VL region of any of the above antibodies (e.g., such as ATL5895, ATL5901 or 5567).

[0358] The overall percent identity of the variable region or full-length heavy / light chain sequences can be combined with specific CDR sequences from the same antibody.

[0359] The antibodies of the present disclosure may comprise one or more substitutions within the framework of the VH and / or VL regions. As used herein, "substitution" refers to the replacement of one amino acid at a specific position with another amino acid relative to the same position in the baseline molecule. In some embodiments, the baseline molecule is an antibody exemplified herein, such as ATL_5331; ATL_5334, or ATL_5335.

[0360] In some embodiments, the antibodies of the present disclosure comprise one or more VH framework substitutions at positions selected from the group consisting of: positions 72, 73, 76, 77, 78, 82A, 82B, 83, 86, and 87 of the VH domain, according to Kabat numbering. In some embodiments, the substitutions are selected from the group consisting of: D72E, D73E, N76A, T77A, V78L, N82AA, S82BT, S87BA, N83K, D86E, and T87A. In some embodiments, the VH framework substitutions are selected from the group consisting of: 78L, 83K, and 76A. In some embodiments, the VH framework substitutions are: (i) 78L and 83K, or (ii) 76A, 78L, and 83K.

[0361] In some embodiments, the VL substitution is at a position selected from the group consisting of: 8, 19, 36, 42, 46, 47, 60, 69, 75, 80, 104. In some embodiments, the substitution is selected from the group consisting of R8A, I19V, F36Y, N42K, P46L, I47M, A60N, N69A, V75I, P80A, and V104L.

[0362] The sequences and properties of the antibodies of the present disclosure can be compared to a "reference antibody." As used herein, a reference antibody is an antibody that binds to the same target as an antibody of the present disclosure but differs in one or more physical properties. For example, a reference antibody may differ in at least one amino acid residue in CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VH framework, VL framework, heavy chain backbone, light chain backbone, Fc region, and / or hinge region, as long as they bind to the same target, preferably the same epitope, as an antibody of the present disclosure. The reference antibody may be isotype-matched to an antibody of the present disclosure. The reference antibody may bind to the same epitope as an antibody of the present disclosure, or may block, sterically hinder, or otherwise compete with an antibody of the present disclosure for the same epitope. The reference antibody may be known in the art, or may have CDRs and / or variable domains of an antibody of the art, but otherwise be identical to an antibody of the present disclosure. For example, ATL_5059 is a reference antibody (which does not have the same CDRs or framework regions as the antibodies of the present disclosure) and is also disclosed as "NI-302.8F1" in US 1 1 ,401 ,325 B2.

[0363] Preferred antibodies have one or more residues that are different from a reference antibody that is capable of binding to the same target and have one or more improved properties relative to the reference antibody. The differences may be in the CDRs and / or framework residues of the variable domains. In some embodiments, the antibody differs from the reference antibody in its CDRs and can bind to the same target, optionally at the same or similar epitope. For example, an antibody according to the present invention may exhibit improved binding efficacy to exon 1 HTT, such as improved binding efficacy to WT HTT and / or improved binding efficacy to mHTT. Without the use of hindsight, it is not easy to determine which residues (if any) in the antibody can improve one or more properties. This can be achieved by obtaining a "parent" reference antibody and striving to change a single residue or a combination of residues and analyzing the results. Alternatively, analysis of variants in a family of naturally occurring antibodies, such as to determine candidate substitutions or phage display analysis of antibodies, can be performed to guide design and collate to produce improved antibodies. Candidate substitutions can be determined from multiple sources and combined for further testing to produce even more favorable antibodies. The antibodies of the present disclosure are determined independently of any prior art antibodies by analyzing the convergence of the B cell repertoire of recovering individuals, or are derived from such antibodies. Therefore, the antibodies of the present disclosure are advantageously derived from naturally occurring protective antibodies, the sequences of which cannot be obtained based on any disclosure in the prior art. For example, only through the important research and guided design described herein were the inventors able to generate antibodies ATL_5895, ATL_5901, and ATL_5667, which are derived from ATL_5331, ATL_5334, and ATL_5335, respectively.

[0364] In some embodiments, the isolated antibody or antibody fragment thereof that specifically binds to a huntingtin (HTT) protein or fragment thereof comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14), or an amino acid sequence comprising an amino acid substitution compared to KAWMN (SEQ ID NO: 14), optionally wherein the substitution is at position 35, optionally wherein the substitution is N35S, wherein position numbering is according to Kabat; ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), or an amino acid substitution compared to RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 16). NO: 15); optionally wherein the substitution is at a position selected from position 64, 54 or 53, optionally wherein the substitution is selected from: Q64K, D53E and G54A, wherein the position numbering is according to Kabat; and iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3), or a sequence comprising one, two, three or four substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3), optionally wherein the substitution is at a position selected from: 95, 97, 100A, 100B, 100C, optionally wherein the substitution is selected from: Y97F, P95S, Y100AG, G100BL and L100C, wherein the position numbering is according to Kabat.

[0365] The isolated antibody or antibody fragment thereof may further comprise a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: iv. LCDR1 has an amino acid sequence selected from the group consisting of TGTSSDVGSYNLVS (SEQ ID NO: 4), TGTSSDVGGYKLVS (SEQ ID NO: 9), and TGTSSDIGSYNLVS (SEQ ID NO: 16); v. LCDR2 has an amino acid sequence selected from the group consisting of EVNKRPS (SEQ ID NO: 5), EVSKRPS (SEQ ID NO: 10), and EGSKRPS (SEQ ID NO: 17); and vi. LCDR3 has an amino acid sequence selected from the group consisting of GSYAGTNNV (SEQ ID NO: 21); and vi. LCDR4 has an amino acid sequence selected from the group consisting of GSYAGTNNV (SEQ ID NO: 22). NO: 21), optionally wherein the substitutions are selected from positions 92, 95, 89 and 91, optionally wherein the substitutions are selected from the group consisting of: A92G, N95A, G89V and Y91F; CSYAGSSVV (SEQ ID NO: 23); an amino acid sequence comprising one or two amino acid substitutions compared to CSYAGSSVV (SEQ ID NO: 23), optionally wherein the amino acid substitutions are at a position selected from the group consisting of: 89, 95, optionally wherein the substitutions are selected from the group consisting of: C89S, N95A; SSYAGFNTLV (SEQ ID NO: 25); and an amino acid sequence comprising one or two amino acid substitutions compared to SSYAGFNTLV (SEQ ID NO: 25), optionally wherein the amino acid substitutions are at positions N95 and / or T95, optionally wherein the substitutions are selected from the group consisting of: N95S and / or T95A, wherein the position numbering is according to Kabat.

[0366] In a related embodiment, the antibody comprises: a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, and optionally a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2 and LCDR3, wherein CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are CDRs comprising one to ten substitutions compared to the following CDRs: i. a HCDR1 having the amino acid sequence KAWMN (SEQ ID NO: 14) or KAWMS (SEQ ID NO: 1), or an amino acid sequence comprising one amino acid substitution compared to KAWMN, optionally wherein the sequence of HCDR1 comprises one substitution compared to KAWMN (SEQ ID NO: 14) or KAWMS (SEQ ID NO: 1);

[0367] ii. a HCDR2 having the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2), RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27), or, optionally wherein the sequence of HCDR2 comprises one, two, three, four, five or six substitutions compared to RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2) or RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27);

[0368] iii. a HCDR3 having the following amino acid sequence: PPYYYYYGLDV (SEQ ID NO: 3), optionally wherein the sequence of HCDR3 comprises one, two, three or four substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3);

[0369] iv. LCDR1 having the following amino acid sequence: TGTSSDVGSYNLVS (SEQ ID NO: 4), TGTSSDVGGYKLVS (SEQ ID NO: 9) or TGTSSDIGSYNLVS (SEQ ID NO: 16), optionally wherein the sequence of LCDR1 comprises one or two substitutions compared to TGTSSDVGSYNLVS (SEQ ID NO: 4), TGTSSDVGGYKLVS (SEQ ID NO: 9) or TGTSSDIGSYNLVS (SEQ ID NO: 16);

[0370] v. LCDR2 having the following amino acid sequence: EVNKRPS (SEQ ID NO: 5), EVSKRPS (SEQ ID NO: 10), or EGSKRPS (SEQ ID NO: 17);

[0371] vi. LCDR3 having the following amino acid sequence:

[0372] GSYAGTNNV (SEQ ID NO: 21), GSYAGTANV (SEQ ID NO: 6), CSYAGSSVV (SEQ ID NO: 23), SSYAGSSVV (SEQ ID NO: 11), SSYAGFSTLV (SEQ ID NO: 18) or SSYAGFNTLV (SEQ ID NO: 25), optionally wherein the sequence of LCDR3 comprises one, two or three substitutions compared to GSYAGTNNV (SEQ ID NO: 21), GSYAGTANV (SEQ ID NO: 6), CSYAGSSVV (SEQ ID NO: 23), SSYAGSSVV (SEQ ID NO: 11), SSYAGFSTLV (SEQ ID NO: 18) or SSYAGFNTLV (SEQ ID NO: 25).

[0373] In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCD2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); and iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3). In some such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6). In other embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9); ii. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10); and iii. LCDR3 has the amino acid sequence SSYAGSSVV (SEQ ID NO: 11).

[0374] In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCD2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14); ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3). In some such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 comprises the amino acid sequence GSYAGTNNV (SEQ ID NO: 21). In other embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9); ii. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10); and iii. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23). In other embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); ii. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17); and iii. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25). In other embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); ii. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17); and iii. LCDR3 has the amino acid sequence SSYAGFSTLV (SEQ ID NO: 18).

[0375] In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCD2, and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3). In some such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence GSYAGTNNV (SEQ ID NO: 21). In other embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9); ii. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10); iii. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23). In other embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); ii. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17); iii. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25).

[0376] In some embodiments, the antibody comprises: a heavy chain variable (VH) domain comprising CDRs HCDR1, HCD2 and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3), or SPYYYYYGLDV (SEQ ID NO: 157), or PPFYYYYGLDV (SEQ ID NO: 158), or PPYYYYGLNV (SEQ ID NO: 159). In some such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4) or TGTSSDVGGYKLVS (SEQ ID NO: 9); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); or EVSKRPS (SEQ ID NO: 10); iii. LCDR3 has the amino acid sequence SSYAGSSVV (SEQ ID NO: 11), or VSFAGTANV (SEQ ID NO: 160), or VSYAGTANV (SEQ ID NO: 161), or VSYGGTENV (SEQ ID NO: 162). In other such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9); ii. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10); iii. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23). In other such embodiments, the antibody comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); ii. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17); iii. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25).

[0377] The VH domain may be a human VH domain. The antibody or fragment thereof may have a VH domain framework sequence selected from the following: (a) the framework sequence of [ATL_0005331VH]: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-CDRH1-WVRQAPGKGLEWVG (SEQ ID NO: 9g)-CDRH2-RFTISRDDSKNTVYLQMNSLNTEDTAVYYCIP (SEQ ID NO: 100)-CDRH3-WGQGTTVTVSS (SEQ ID NO: 101), (b) the framework sequence of [ATL_0005334VH]: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-CDRH1-WVRQAPGKGLEWVG (SEQ ID NO: 99)-CDRH2-RFTISRDDSKNTVYLQMNSLNTEDTAVYYCIP (SEQ ID NO: 100) (SEQ ID NO: 100)-CDRH3-WGQGTTVTVSS (SEQ ID NO: 101), and (c) the framework sequence of [ATL_0005335 VH]: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-CDRH1-WVRQAPGKGLEWVG (SEQ ID NO: 99)-CDRH2-RFTISRDDSKNTVYLQMNSLNTEDTAVYYCIP (SEQ ID NO: 100)-CDRH3-WGQGTTVTVSS (SEQ ID NO: 101). The antibody or fragment thereof may have a VH domain framework sequence selected from the following: (a) the framework sequence of ATL 0006199 VH:

[0378]

[0379] (b) Framework sequence of ATL_0006200VH (and ATL_0006374):

[0380]

[0381] (c) Framework sequence of ATL_0006202VH:

[0382]

[0383] (d) Framework sequence of ATL_0006203VH:

[0384]

[0385] (e) Framework sequence of ATL_0006205VH (as well as ATL_0006375, ATL_0006376, ATL_0006377, and ATL_0006378):

[0386]

[0387] The isolated antibody or fragment thereof may comprise one or more framework substitutions in the VH domain (e.g., compared to the above framework sequences). The one or more framework substitutions may be located at a position selected from the group consisting of: 72, 73, 76, 77, 78, 82A, 82B, 83, 86, and 87. The one or more framework substitutions may be located at a position selected from the group consisting of: 72, 73, 76, 77, 78, 82A, 82B, 83, 86, 87, 91, and 93. One or more framework substitutions in the VH domain may be selected from: at position 72: E (e.g., D72E), at position 73: E (e.g., D73E), at position 76: A (e.g., N76A), at position 77: A (e.g., T77A), at position 78: L (e.g., V78L), at position 82A: A (e.g., N82AA), at position 82B: T (e.g., S82BT), at position 82B: A (e.g., S87BA), at position 83: K (e.g., N83K), at position 86: E (e.g., D86E), at position 87: A (e.g., T87A), at position 91: W (Y91W), and at position 93: M, S or V (I93M, I93S, I93V); wherein the position numbering is that of Kabat. Such mutations in the framework region can advantageously remove liability, improve the stability of the resulting antibody and reduce its immunogenicity. For example, mutations D72E and / or D73E can reduce the risk of isomerization, N76A and / or T77A can reduce the risk of deamination, D86E and / or T87A can reduce the risk of isomerization, and mutations N82AA, S82BT, N83K and / or S87BA can reduce the risk of deamination. In some embodiments, the framework replacement is selected from: 78L, 83K and 76A, optionally wherein the framework replacement is: (i) 78L and 83K, or (ii) 76A, 78L and 83K.

[0388] In some embodiments, the VL domain is a human VL domain. In some embodiments, the antibody or fragment thereof has a VL domain framework sequence selected from the following: (a) the framework sequence of [ATL_0005331VL]:

[0389] (b) Framework sequence of [ATL_0005334VL]:

[0390]

[0391] and (c) the framework sequence of [ATL_0005335VL]:

[0392]

[0393] In some embodiments, the isolated antibody or fragment thereof comprises one or more framework replacements in the VL domain (e.g., compared to the above framework sequences). The one or more framework mutations may be located at a position selected from the group consisting of: position 8, 19, 36, 42, 46, 47, 60, 69, 75, 80, 104. One or more framework substitutions in the VL domain may be selected from: at position 8: R (e.g., R8A), at position 19: V (e.g., I19V), at position 36: Y (e.g., F36Y), at position 42: K (e.g., N42K), at position 46: L (e.g., P46L), at position 47: M (e.g., I47M or L47M), at position 60: N (e.g., A60N), at position 69: A (e.g., N69A), at position 75: I (e.g., V75I), at position 80: A (e.g., P80A), and at position 104: V or L (e.g., L104V or V104L), where position numbering is according to Kabat.One or more framework mutations may be located at a position selected from the group consisting of: (i) in antibody ATL_0005334, an antibody derived therefrom (e.g., ATL_0005586, ATL_0005900, ATL_0005556, ATL_0005901), or a VL sequence comprising any of ATL_0005586, ATL_0005900, ATL_0005901, ATL_0005556 (e.g., SEDID No: 13, 24): position 8 (optionally wherein the substituted amino acid is R); (ii) in antibody ATL_0005331 or an antibody derived therefrom (e.g., ATL_0005577, ATL_0005891, ATL_0005890, ATL_0005559, ATL_0005563, ATL_0005896, ATL_0005894, ATL_0005331); L_0005895, ATL_0005555), or a VL sequence comprising any one of ATL_0005577, ATL_0005891, ATL_0005890, ATL_0005559, ATL_0005563, ATL_0005896, ATL_0005894, ATL_0005895, ATL_0005555 (e.g., SEQ ID No: 22, 32, 29, 40, 41, 8): position 19 (optionally wherein the substituted amino acid is V), position 36 (optionally wherein the substituted amino acid is Y), position 47 (optionally wherein the substituted amino acid is M), position 69 (optionally wherein the substituted amino acid is A), position 75 (optionally wherein the substituted amino acid is I), position 104 (optionally wherein the substituted amino acid is V); (iii) in antibody ATL_0005335 or an antibody derived therefrom (e.g., ATL_0005572, ATL_0005567, ATL_0005557), or comprising the VL sequence of any one of ATL_0005572, ATL_0005567, ATL_0005557 (e.g., SEQ ID No: 30, 26, 20, 39): position 42 (optionally wherein the substituted amino acid is R), position 46 (optionally wherein the substituted amino acid is K), position 47 (optionally wherein the substituted amino acid is M), position 60 (optionally wherein the substituted amino acid is N), position 69 (optionally wherein the substituted amino acid is A), position 80 (optionally wherein the substituted amino acid is A), position 104 (optionally wherein the substituted amino acid is L). Such mutations in the framework region can advantageously remove susceptibility, improve the stability of the resulting antibody and reduce its immunogenicity. For example, the substitution N69A (or 70A) can reduce the risk of deamination.

[0394] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the VH domain are in germline framework. In some embodiments, the LCDR1, LCDR2, and LCDR3 of the VL domain are in germline framework.

[0395] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0396] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0397] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0398]

[0399] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0400]

[0401] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0402]

[0403] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0404]

[0405] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0406]

[0407] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0408]

[0409] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0410]

[0411] In some such embodiments, the light chain variable domain sequence comprises the amino acid sequence

[0412]

[0413] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0414]

[0415] In some such embodiments, the light chain variable domain sequence

[0416]

[0417] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0418]

[0419] In some such embodiments, the light chain variable domain sequence

[0420]

[0421] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0422]

[0423] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0424]

[0425] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0426]

[0427] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0428]

[0429] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0430]

[0431] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0432]

[0433] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0434]

[0435] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0436]

[0437] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0438]

[0439] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0440]

[0441] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0442]

[0443] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0444]

[0445] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0446] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0447] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0448] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0449] or

[0450] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0451] In some embodiments, the light chain variable domain comprises the amino acid sequence

[0452] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0453] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0454] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0455] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0456] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0457] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0458] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0459] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0460] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0461] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0462] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0463] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0464] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0465] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0466] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0467] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0468] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0469] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0470] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0471] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0472] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0473] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0474] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0475] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0476] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0477] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0478] In some embodiments, the heavy chain variable domain comprises the amino acid sequence

[0479] In some such embodiments, the light chain variable domain comprises the amino acid sequence

[0480]

[0481] In some embodiments, the heavy chain variable domain comprises a variable domain comprising an amino acid sequence having at least 91% sequence identity to any of the above heavy chain variable domains (e.g., SEQ ID Nos: 7, 12, 19, 28, 31). In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% sequence identity to any of the above light chain variable domains (e.g., SEQ ID Nos: 8, 13, 20, 22, 24, 26, 29, 30, 32, 39, 40, 41, 42). Any of the above heavy chains (e.g., SEQ ID Nos: 7, 12, 19, 28, 31) can be combined with any of the above light chains (e.g., SEQ ID Nos: 8, 13, 20, 22, 24, 26, 29, 30, 32, 39, 40, 41, 42).

[0482] In some embodiments, the antibodies of the invention have improved binding potency compared to a reference antibody. The improved binding potency may be one or more residues in the CDR or V H or V L Binding potency and half maximal effective concentration (EC 50 ) value or the concentration required to obtain 50% binding. Binding potency can be measured using an ELISA-based assay as known in the art (eg, HTT sandwich ELISA).

[0483] In some embodiments, the isolated antibody or fragment thereof binds to mHTT and / or aggregated HTT protein (or a fragment thereof, preferably comprising exon 1) as determined by immunoprecipitation (e.g., immunoprecipitation of mHTT and / or aggregated HTT using an antibody or fragment thereof of the disclosure).

[0484] In some embodiments, the antibodies or fragments thereof according to the present disclosure are capable of crossing the blood-brain barrier. In some embodiments, the antibodies are bispecific antibodies. For example, the antibodies according to the present disclosure may have an scFV chain that binds to a receptor in the brain (e.g., transferrin receptor (Yu et al., Sci Transl Med. 2014 Nov 5; 6(261): 261ra154.)), and an scFV chain that binds to HTT as described herein. In some embodiments, the antibodies according to the present disclosure comprise an antibody or fragment thereof (e.g., such as an antibody, scFV, sdAb, etc.) that binds to HTT as described herein, and an additional binding portion that binds to another target. The other target may be a receptor in the brain, such as a transferrin receptor. The additional binding portion may be an antibody, scFv, nanobody, or aptamer. The two binding portions of such a bispecific molecule may form a fusion protein.

[0485] Also described herein are single domain antibodies (sdAbs), also referred to as nanobodies, comprising the heavy chain CDRs and / or VH sequences of any of the antibodies described herein. Thus, also described herein are antibodies or fusion molecules comprising nanobodies that bind to HTT as described herein, and nanobodies that bind to receptors in the brain. Also described herein are antibodies comprising fusion molecules of scFV chains or nanobodies that bind to HTT as described herein, and aptamers that bind to receptors in the brain.

[0486] Provided are isolated nucleic acids encoding antibodies, antigen-binding fragments, or polypeptides as described herein. Also provided are vectors comprising the nucleic acids described herein and host cells comprising the vectors. For example, the host cell can be a eukaryotic cell or a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell), or can be a prokaryotic cell, such as Escherichia coli. In some embodiments, the vector is a viral vector, such as a bacteriophage.

[0487] Also provided are methods for preparing an antibody or antigen-binding fragment or polypeptide as described herein, comprising culturing a host cell as described herein under conditions suitable for expressing a vector encoding the antibody or antigen-binding fragment or polypeptide, and isolating and / or purifying the antibody or antigen-binding fragment or polypeptide. The method further comprises formulating the antibody or antibody fragment into a composition comprising at least one additional component.

[0488] The antibodies and fragments thereof described herein can be used therapeutically.

[0489] The subject to be treated or diagnosed can be any animal or human. The subject is preferably a mammal, more preferably a human. The subject can be male or female. The subject can be a patient. The therapeutic use can be in humans or animals (veterinary use).

[0490] Medicaments and pharmaceutical compositions according to aspects of the present invention can be formulated for administration by a variety of routes including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, oral, and nasal.Medicaments and compositions can be formulated for injection.

[0491] Pharmaceutical compositions can be prepared using pharmaceutically acceptable "carriers" consisting of substances that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., molecular entities and compositions that are physiologically tolerable and generally do not produce allergic reactions or similar adverse reactions, such as stomach discomfort, when administered to humans. In some embodiments, the term refers to molecular entities and compositions approved by regulatory agencies of the U.S. federal or state governments, such as the GRAS listing under Sections 5, 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, which undergo pre-market review and approval by the FDA or similar lists, the U.S. Pharmacopeia, or other generally recognized pharmacopeias for use in animals and more specifically in humans. The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and methods of using such carriers to compound pharmaceutical compositions.

[0492] The pharmaceutical compositions provided herein may include one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonic agents, antioxidants, or antimicrobial preservatives. When used, the excipients in the composition will not adversely affect the stability, bioavailability, safety, and / or effectiveness of the active ingredient (i.e., the anti-CFH antibody used in the composition). Therefore, it will be understood by those skilled in the art that compositions are provided in which there is no incompatibility between any components of the dosage form. Excipients may be selected from buffers, solubilizers, tonicity agents, chelating agents, antioxidants, antimicrobials, and preservatives.

[0493] It is preferably administered in a "therapeutically effective amount" sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. Treatment prescriptions (e.g., dosage determinations, etc.) are within the purview of general practitioners and other physicians, and generally take into account the condition to be treated, the condition of the individual patient, the delivery site, the method of administration, and other factors known to the physician. Some examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th edition, 2000, pub. Lippincott, Williams & Wilkins.

[0494] Treatable conditions according to the present disclosure include any condition in which HTT plays a role, including neurodegenerative conditions, and in particular those characterized by pathological accumulation of abnormal protein aggregates in the brain (e.g., aggregation of mHTT). Treatable conditions according to the present disclosure include any polyQ (polyglutamine)-related diseases (see, e.g., Cell Transplant. 2014; 23(4-5): 441-58). PolyQ diseases are neurodegenerative conditions caused by CAG repeat expansion in specific proteins. They include: six spinocerebellar ataxias (SCA) 1, 2, 6, 7, 17; Machado-Joseph disease (MJD / SCA3); Huntington's disease (HD); dentatorubralpallidoluysian atrophy (DRPLA); and X-linked spinal bulbar muscular atrophy type 1 (SMAX1 / SBMA). The antibodies of the present disclosure were initially identified through analysis of patients recovering from Alzheimer's disease and have shown relevance to other aggregation-related neurodegenerative diseases in addition to HD. The neurodegenerative disease or disorder may include one or more of the following: Huntington's disease; Alzheimer's disease (AD); frontotemporal dementia; Parkinson's disease (PD); amyotrophic lateral sclerosis (ALS); prion disease; Lewy body disease; spinal muscular atrophy (SMA); motor neuron disease (MND); progressive supranuclear palsy (PSP); spinocerebellar ataxia types 1, 2, 6, 7, and 17 (SCA); Machado-Joseph disease (MJD / SCA3); dentate-rubral-pallidothalamic atrophy (DRPLA); X-linked spinal bulbar muscular atrophy type 1 (SMAX1 / SBMA); Anderson-Fabry (X-linked Fabry disease); Disease); and DNAJB6 myopathy.

[0495] The antibodies of the present disclosure can be used in combination with additional therapeutic agents for treatment. As used herein, an "additional therapeutic agent" is another compound, protein, vector, antibody, cell, or entity that has a therapeutic effect. The antibodies can be co-administered with the additional therapeutic agent. The antibodies can be co-formulated with the additional therapeutic agent. The antibodies can be administered sequentially before or after the additional therapeutic agent.

[0496] In some embodiments, the antibodies and fragments thereof described herein can be used in methods for diagnosing or monitoring the progression of a disease or condition characterized by the presence of a mutated or aggregated HTT protein in a patient. The presence of mutated and / or aggregated HTT indicates the progression of the disease. The level of HTT can be quantified in the cerebrospinal fluid or blood and samples derived from the patient. The level of mutated and / or aggregated protein can be quantified using any technique known in the art. A variety of assays are available, including ELISA, flow cytometry, and Western blotting. Therefore, also described herein is a method for detecting the presence and / or amount of mutated or aggregated HTT protein in a sample (e.g., a sample obtained from a patient diagnosed with or suspected of having a disease or condition characterized by the presence of a mutated or aggregated HTT protein), the method comprising using an antibody or fragment thereof as described herein (e.g., to label, separate, etc. the presence of mutated or aggregated HTT in the sample).

[0497] The antibodies described herein can be used as biomarkers to indicate that a subject may have or develop a disease or condition characterized by the presence of a mutated or aggregated HTT protein. For example, a method of diagnosing a disease or condition characterized by the presence of a mutated or aggregated HTT protein in a patient may comprise obtaining BCR sequence data from the subject and using the sequence data to determine whether the subject's BCR repertoire contains one or more antibodies that may bind to HTT (e.g., antibodies as described herein, such as antibodies that are at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to any specific antibody or antibody fragment described herein), wherein a subject whose BCR repertoire contains one or more antibodies that may bind to HTT may have or be at risk for developing a disease or condition characterized by the presence of a mutated or aggregated HTT.

[0498] The antibodies described herein can be used as biomarkers indicating that a subject may be responsive to treatment with an antibody or antibody fragment as described herein. A method for determining whether a subject may be responsive to treatment with an antibody or antibody fragment as described herein, the method comprising obtaining BCR sequence data from a subject and using the sequence data to determine whether the subject's BCR library contains one or more antibodies that may bind to HTT (e.g., an antibody as described herein, such as an antibody having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to any specific antibody or antibody fragment described herein), wherein a subject whose BCR library does not contain one or more antibodies that may bind to HTT may be responsive to treatment with an antibody or antibody fragment as described herein. Thus, also described herein are methods of treating a subject diagnosed as having or at risk for a disease or disorder associated with HTT and / or polyQ aggregation (e.g., a neurodegenerative disorder), the methods comprising: obtaining BCR sequence data from the subject; using the sequence data to determine whether the subject's BCR repertoire comprises one or more antibodies that may bind to HTT; and administering a therapeutically effective amount of an antibody or antibody fragment thereof as described herein to the subject whose BCR repertoire does not comprise one or more antibodies that may bind to HTT.

[0499] Some methods of the present disclosure relate to samples containing cells. The sample can be a cell culture grown in vitro. For example, the culture can contain cells or a suspension of cells cultured in a culture plate or culture dish. The methods according to the present disclosure can be performed in vitro, ex vivo or in vivo, or the products can be present in vitro, ex vivo or in vivo. The term "in vitro" is intended to cover experiments performed under laboratory conditions or in culture using materials, biological substances, cells and / or tissues, while the term "in vivo" is intended to cover experiments and operations performed using intact multicellular organisms. "Ex vivo" refers to something that exists or occurs outside an organism (e.g., outside the human or animal body), which can be on a tissue (e.g., a whole organ) or cell taken from an organism.

[0500] According to some aspects of the present disclosure, a kit comprising an antibody according to the present invention is provided. In some embodiments, the kit comprises an antibody according to the present invention and one or more of the following: reagents for immunochemistry; antibodies immobilized to a solid support; means for labeling the antibody; means for linking the antibody to a cytotoxic moiety; and additional therapeutic agents.

[0501] Percentage (%) sequence identity is defined as the percentage of the amino acid residues in the candidate sequence that are identical with the residue in the comparison sequence after aligning the sequences and introducing a gap (gap) (if necessary) to achieve maximum sequence identity and without considering any conservative substitution as a part for sequence identity. Sequence identity is preferably calculated over the entire length of each sequence. When the compared sequences have different lengths, the sequence identity of the shorter comparison sequence can be determined over the entire length of the longer given sequence, or when the comparison sequence is longer than the given sequence, the sequence identity of the comparison sequence can be determined over the entire length of the shorter given sequence. Sequence identity can be defined with reference to algorithm GAP (Wisconsin GCG bag, Accelerys Inc, San Diego USA). GAP uses Needleman and Wunsch algorithm to compare two complete sequences, which maximizes the number of matches and minimizes the number of gaps. Generally speaking, default parameters can be used, wherein gap generation penalty=12 and gap extension penalty=4. The use of GAP may be preferred, but other algorithms may also be used, such as BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), SSEARCH (Smith and Waterman (1981) J. Mol Biol. 147:195-197;), HMMER3 (Johnson LS et al BMC Bioinformatics. 2010 Aug 18;11 ():431), or the TBLASTN program (Altschul et al. (1990) supra), typically using default parameters (see, for example, Pearson Curr Prot Bioinformatics (2013) Chapt 3 Uniy3.1 doi: 10.1002 / 0471250953.bi0301s42). In particular, the psi-Blast algorithm (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402) can be used. Sequence identity and similarity can also be determined using Genomequest TMSoftware (Gene-IT, Worcester MA USA) is used to determine the sequence comparison. Sequence comparisons are preferably performed over the entire length of the relevant sequences to be compared. Features disclosed in the foregoing description, in the appended claims, or in the accompanying drawings in their specific form or in terms of the manner in which they are used to perform the disclosed functions, or the methods or processes for obtaining the disclosed results, may be used to implement the present invention in its various forms, either individually or in any combination of such features, as appropriate.

[0502] Although the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art upon giving this disclosure. Therefore, the exemplary embodiments of the present invention set forth above are intended to be illustrative and not restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

[0503] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0504] Throughout this specification (including the appended claims), unless the context requires otherwise, the words "comprise" and "include" and variations thereof will be understood to imply the inclusion of the indicated whole or step or group of wholes or steps, but not to the exclusion of any other whole or step or group of wholes or steps. It must be noted that, unless the context clearly indicates otherwise, nouns as used in the specification and the appended claims without quantifiers include plural referents. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one said particular value and / or to another said particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means, for example, + / - 10%.

[0505] All references cited herein are hereby incorporated by reference in their entirety.For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3d. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0506] sequence

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521] Table 1. Sequences mentioned in this disclosure. Note that the "ATL_" identifier is the antibody identifier, and the "AC_" identifier refers to unique individual chains, some of which are shared between different antibodies.

[0522] Example

[0523] The following examples illustrate the identification and characterization of exemplary HTT antibodies of the present disclosure.

[0524] Materials and Methods

[0525] Capillary isoelectric focusing

[0526] Charge variant analysis was performed on the lead antibody by preparing a master mix to dilute the antibody sample to run on a cIEF cartridge on a Maurice instrument (Protein Simple). The master mix had the following final concentrations: methylcellulose 0.35%, pharmalyte 3-10 4%, 10 mM arginine, and pI markers 4.09 and 9.99 0.01%. The sample was diluted in the master mix at 0.15 to 0.25 mg / ml and run at 1500 volts for 1 minute followed by 3000 volts for 4.5 minutes. A system suitability standard (Protein Simples) was also run at the beginning and end of the run. Data generated from the same sample subjected to stress conditions were superimposed to compare charge species profiles.

[0527] 5×FT cycle

[0528] 5 mg / ml mAb samples were stored at -80°C and frozen and thawed for 5 cycles over an 8-hour period at room temperature (RT). The final sample after 5 freeze-thaw cycles was diluted to 0.7 mg / ml for purity analysis on SEC-HPLC.

[0529] SEC-HPLC

[0530] Antibody samples were diluted to 0.7 mg / ml in 20 mM histidine acetate, 150 mM NaCl pH 5.5 for running on a Zorbax GF-250 SEC-HPLC column (Agilent) on a Vanquish Flex (Thermo). Samples were size separated in a mobile phase of 20 mM sodium phosphate, 300 mM sodium sulfate, and 100 mM arginine at a flow rate of 0.75 ml / min at 25°C for 25 minutes per sample. Chromatograms were integrated using Chromeleon software (Thermo). Recombinant HTT exon-1 binding ELISA

[0531] An indirect ELISA was performed using two different constructs of recombinant HTT exon-1 with a His-tag and a GST-tag (WT human HTT exon 1 25Q GST and WT human HTT exon 1 48Q GST) and two different constructs of recombinant HTT exon-1 with only a His-tag (WT human HTT exon 1 25Q His and WT human HTT exon 1 48Q His). Each construct or an irrelevant protein lysozyme as a control was coated onto a Nunc Maxsorp plate at 5 ug / ml in PBS and incubated overnight at 4°C. The plate was blocked with PBS + 2% skim milk powder for 2 hours at room temperature. The buffer was discarded and the plate was washed three times with PBS + 0.1% Tween-20. The test or control antibody was added to the wells at 100 ug / ml in PBS + 2% skim milk powder and incubated at room temperature for 1 hour. The buffer was discarded and the plate was washed three times with PBS + 0.1% Tween-20. An HRP-conjugated secondary antibody in PBS + 2% skim milk powder was added to the plate and incubated at room temperature for 30 minutes. The buffer was discarded and the plate was washed three times with PBS + 0.1% Tween-20. TMB substrate was added to each well and allowed to develop at room temperature for 5-10 minutes, followed by termination with 0.2 M NaOH. The plate was read, measuring absorbance at 450 nm.

[0532] Thermal shift measurement

[0533] Protein thermal shift measurements were performed on a QuantiStudio 5 real-time qPCR (ThermoFisher). Antibodies were diluted to 0.1 to 0.25, with 10× Sypro orange protein gel stain (Thermo# S6651) added. Samples were loaded into a 384-well Microamp qPCR plate. Samples were run through a temperature range of 25 to 95°C at 2-minute intervals. Protein melting curves were analyzed using protein thermal shift software to determine Tm values, which correlate with antibody stability.

[0534] HTT sandwich ELISA

[0535] To evaluate the binding potency of the antibodies to the HTT protein, the binding of the antibodies to HTT exon 1 with 25Q repeats or 48Q repeats (for sequences, see "Sequence") was evaluated in a sandwich ELISA format and the EC50 was calculated. Anti-HTT capture antibody (Merck Millipore; #MABN2427) was diluted to 4.17 μg / ml in 1× ELISA coating buffer (Biolegend; #421701). 50 μl was added to each well of a 96-well plate and placed at 4°C overnight. The plate was washed with PBS / 0.1% Tween. The plate was blocked with 50 ul / well of blocking buffer (1% BSA / PBS). The plate was washed with PBS / 0.1% Tween. 50 μl of diluted target antigen (HTT exon 1 48Q GST or HTT exon 1 25Q GST) and lysozyme (negative antigen control) were added to the plate at 0.04 ug / ml. The plate was incubated on a plate shaker (300 rpm) at RT for 1 hour. The plate was washed with PBS / 0.1% Tween. An 8-point 3-fold serial dilution of the test antibody (usually starting at 400 nM) was added to the plate. Another set of wells received only 50 μl / well of buffer (blank control). The plate was incubated on a plate shaker (300 rpm) at RT for 1 hour. The plate was washed with PBS / 0.1% Tween. 50 μl of anti-human IgG HRP (80 ng / ml; Jackson ImmunoResearch; #109-035-097; Lot: 160716) was added to each well. The plate was washed with PBS / 0.1% Tween and 50 μl of TMB solution (Lifetechnology; #002023) was added. Incubated at RT for 6 minutes in the dark. 50 μl of stop solution was added to the plate (Fither chemical, #12933634). The absorbance at 450 nm was read on a CLARIO Star. Background (average of wells without test antibody) was subtracted from all values. EC50 (half maximal concentration) values were calculated using a nonlinear four-parameter curve fit without restriction.

[0536] In vivo PK studies

[0537] To determine the serum PK of the lead antibody, an in vivo PK experiment was performed using ATL_0005335 (the parent antibody of ATL_0005567). Female 6-8 week old C57BL6J mice received 10 and 20 mg / kg of ATL_0005335 by IP injection and serum was collected 1, 4, 8, 24, 72, and 144 hours after administration. Antibody levels were assessed by ELISA of serum samples.

[0538] HTT immunoprecipitation of U-2OS cell lysate

[0539] Using Protein G-coated Dynabeads TM Immunoprecipitation was performed using a magnetic rack (Thermosfiher; 10014D). After incubation with the antibody, the beads were washed using a magnetic rack and incubated with U-2OS cell lysates (from either the parental form or the HTT 110CAG expression form). The beads were washed again and the captured proteins were eluted and analyzed by automated western blotting (Bio-techne; Jess). Detection was performed using the anti-HTT antibody 1C2 (Merck / Millipore MAB1574).

[0540] HTT immunoprecipitation using R6 / 2 mouse brain

[0541] Using Protein G-coated Dynabeads TM Immunoprecipitation was performed using a magnetic stand (Thermofisher; 10014D). After incubation with the antibody, the beads were washed using a magnetic stand and incubated with brain homogenates from R6 / 2 mice or non-transgenic (non-Tg) littermates. The beads were washed again and the captured proteins were then eluted and analyzed by western blotting. Detection was performed using anti-HTT antibodies MW8 (Merck / Millipore MABN2529) and 1C2 (MAB1574 Sigma-Aldrich). The proteins were expressed by ATL_0005895, ATL_0005901, and ATL_0005567 ( Figure 17B ) and ATL_0005335 (data not shown) successfully immunoprecipitated high molecular weight HTT species.

[0542] HTT immunoprecipitation using human Huntington's disease brain

[0543] Brain homogenates were prepared from superior temporal gyrus tissue from postmortem brains of Huntington's disease patients. Homogenates were prepared in BLB (Brain Lysis Buffer: 10 mM Tris-HCl pH 7.4, 0.8 M NaCl, 1 mM EDTA, 10% sucrose) containing benzonase and protease inhibitors. The homogenates were centrifuged at 2,700 × g and the supernatant was used for IP. Protein G-coated dynabeads were coupled to isotype control antibodies or ATL_0005895 and the brain homogenates were then IPed in brain lysis buffer overnight at 4°C on a rotating wheel. On the next day, the beads were washed in BLB, then heat-denatured in western blot sample buffer and analyzed by western blotting. Detection was performed with anti-HTT antibodies HD1 and MW1 (MABN2427 Millipore).

[0544] Phagocytosis assay

[0545] To monitor phagocytic activity in the cultures, latex beads coated with Q48HTT (Invitrogen, 11564067) were stained with a pH-sensitive pHrodo TM Red succinimidyl ester (Thermo Fisher, P36600) was used for labeling. Microglia derived from induced pluripotent stem cells (iPSC) (Fujifilm, C1110) were seeded in 96-well plates at 23,000 cells / well and left to stand for 3 days, with 50% culture medium replacement on the 3rd day. On the 4th day, 0.5% latex bead solution was incubated with antibodies at 1, 2.1, 4.2, 8.3, 16.7, 33.3, 133.3, 266.7 and 333.3 (nM) and added to microglia at a dilution of 1 / 80. Phagocytosis was monitored for 4 hours by Incucyte (Sartorius).

[0546] Phagocytosis of the beads induces a red fluorescent signal in response to the intracellular environment with a low pH. The change in the total area of the red fluorescent signal over time indicates the rate of phagocytosis and the amount of bait ingested. To evaluate the effect of ATL_0005895 (also referred to herein as ATLX_1095) on phagocytosis, the bead-48QHtt-pHrodo™ Red complex was incubated with ATL_0005895 or a human IgG1 isotype control antibody for 1 hour, and then the cells were exposed to the antibody-treated bead-48QHtt-pHrodo™ Red complex.

[0547] In vivo PK assay

[0548] To determine the serum PK of selected HTT antibodies, in vivo PK experiments were performed using 10 mg / kg of ATL_5567 and ATL_5901. Male 6-8 week old C57BL6J mice were injected intraperitoneally (IP) with 10 mg / kg of the relevant antibody and serum was collected 1, 4, 8, 24, 72, and 144 hours after administration. Antibody levels were assessed by ELISA of serum samples.

[0549] ATL-5895 was tested at 1, 10, and 60 mg / kg to determine serum and CSF PK. Male 6- to 8-week-old C57BL6J mice received 10 mg / kg of the relevant antibody via IP injection. Serum was collected 1, 4, 8, 24, 72, and 144 hours after administration. CSF was collected 4 and 144 hours after administration.

[0550] In vivo PD assay

[0551] To evaluate the effect of ATL_5895 on HTT aggregate load in R6 / 1 mice, an in vivo PD experiment was performed using 60 mg / Kg ATL_5895. The mice used were mixed-sex R6 / 1 (Jackson Laboratory Stock No: 006471). They were 5 weeks old at the start of the study and were treated for a maximum of 12 weeks (17 weeks old). Non-Tg littermates were used as wild-type controls. Mice were administered once a week via IP with 60 mg / Kg ATL_5895 or vehicle control (histidine acetate buffer). Tissue samples were collected after 0, 4, 8, and 12 weeks of treatment. Samples for HTT analysis were quickly frozen in liquid nitrogen and stored at -80 degrees Celsius until analysis. For analysis, samples were prepared into lysates in MSD lysis buffer supplemented with NaF, PMSF, protease inhibitor cocktail 1 (Mini, EDTA-free, Cat#04693159001, Roche) 2 (Sigma, Cat.#P5726) and 3 (Sigma, Cat.#P0044). Aggregated HTT from striatum and cortex samples was then analyzed by mesoscale discovery (MSD) using the 4C9 / MW8 antibody pair; soluble mutant HTT levels were determined by MSD using the 2B7 / MW1 antibody pair; and mouse endogenous HTT levels were determined by MSD using the 2B7 / D7F7 antibody pair. MW1 (MABN2427 Sigma), MW8 (MABN2529 Sigma), 4C9 (Coriell CH03157) and 2B7 (Coriell CH03023) are anti-HTT mouse monoclonal antibodies. D7F7 is an anti-HTT rabbit monoclonal antibody. Statistical significance was assessed using the Mann Whitney test.

[0552] Phage display

[0553] Phage libraries of scFv sequences derived from ATL_5895 were generated. These libraries contained: (1) VH sequences that were softly randomized in CDR3H (I105 to V117—IMGT numbering, corresponding to 93 to 102 Kabat) or hard randomized in Y103 (Y91), I105 (I93), P106 (P94 Kabat), G114 (G100B), and L115 (L100C) (IMGT numbering, Kabat in parentheses), and (2) CDR3L soft randomized variants (randomized in the following: G105 to V117—IMGT numbering, corresponding to 89 to 97 Kabat) or VL of ATL_5895. Soft randomization was performed using degenerate oligonucleotides synthesized from a 70-10-10-10 mixture of nucleotide bases, with an excess of original (ATL_5895) nucleotides. Hard randomization was performed with degenerate oligonucleotides with NNS codons.

[0554] For each library, 3 rounds of phage display were performed. Phage display was performed for fixed human HTT exon 1 48Q GST protein or biotinylated GYSLPQPQPPPPPPPPPP peptide in solution. The phage library was incubated with antigen and then washed to remove unbound phage. Bound phage was then eluted using trypsin (selection) or IgG elution buffer (biotinylated peptide selection). TG1 cells were infected with the eluted phage and subsequently plated on selective culture medium.

[0555] Colonies from rounds 2 and 3 were sequenced and subjected to phage ELISA against at least one antigen to evaluate binding of phage clones. Sequences with improved binding in phage ELISA relative to ATL_5895 were expressed in IgG1 format.

[0556] Indirect ELISA

[0557] An indirect ELISA was performed using HTT exon-1 48Q GST. An irrelevant protein, lysozyme, was used as a control. Each construct was coated onto Nunc Maxisorp plates at 5 μg / ml in PBS and incubated overnight at 4°C. The plates were blocked with PBS + 3% skim milk powder for 1 hour at room temperature. The buffer was discarded and the plates were washed three times with PBS + 0.1% Tween-20. An 8-point 3-fold serial dilution of the test or control antibody (starting at 60 μg / ml) was added to the wells in PBS + 3% skim milk powder and incubated for 1 hour at room temperature. For each antigen, one well received 50 μl of buffer only (blank control). The plates were incubated for 1 hour at room temperature. The buffer was discarded and the plates were washed three times with PBS + 0.1% Tween-20. An HRP-conjugated secondary antibody in PBS + 2% skim milk powder was added to the plates and incubated for 1 hour at room temperature. The buffer was discarded and the plates were washed three times with PBS + 0.1% Tween-20. TMB substrate was added to each well and allowed to develop for 2 minutes at room temperature. The reaction was terminated with 0.5% sulfuric acid. The absorbance at 450 nm was read for each well.

[0558] Seeding assay

[0559] To determine the ability of the antibodies to bind to seed competent HTT species and affect HTT aggregation, the aggregation rate of FRET-labeled recombinant HTT was assessed by FRASE assay as described in Ast et al. (mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease, Molecular Cell, Vol. 71, Issue 5, P675-688. E6, Sept 06, 2018—incorporated herein by reference). Briefly, soluble glutathione S-transferase (GST) HTT exon-1 (HTTex1) fusion proteins with 48 glutamines fused to CyPet or YPet at the C-terminus (GST-Ex1Q48-CyPet or GST-Ex1Q48-YPet) were produced in E. coli BL21-CodonPlus-RPBL21-CodonPlus-RP and affinity purified on glutathione-Sepharose beads. The purified protein was dialyzed overnight at 4°C against 50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA and 5% glycerol, flash frozen in liquid N2 and stored at -80°C.

[0560] R6 / 2 frozen brain tissue was cut on dry ice, weighed, and homogenized using a dounce homogenizer in a 10-fold excess (w / v) of ice-cold 10 mM Tris-HCl pH 7.4, 0.8 M NaCl, 1 mM EDTA, 10% sucrose, 0.25 U / μl benzonase, and a complete protease inhibitor cocktail. The homogenate was incubated at 4°C on a rotator for 1 hour and centrifuged at 2,700 × g (4°C) for 20 minutes to remove cell debris.

[0561] The two recombinant Ex1Q48-CyPet and -Ex1Q48-YPet proteins were cleaved with PreScission protease (PSP) to release GST and initiate spontaneous aggregation of the fusion proteins Ex1Q48-CyPet and Ex1Q48-Ypet. This aggregation resulted in a time-dependent and concentration-dependent increase in FRET. Aggregation was tested in the presence of 10 nM fibrils produced by recombinant HTT and 2.5 μg brain homogenate from R6 / 2 mice. Immunodepletion of seeds using antibodies ATL_0005895 and ATL_0005901 of the present disclosure, as well as MW8 (Millipore; MABN2529) and MW1 (Millipore; MABN2427) was tested. 25 μl of protein G beads (lifetechnologies) and 6 μg of antibody were used for immunodepletion.

[0562] In vitro selectivity assay

[0563] To assess the selectivity of the antibodies, ATL_5895, ATL_5901, and ATL_5567 were screened for binding to fixed HEK293 cells expressing 6105 individual full-length human plasma membrane proteins, secreted and cell surface-tethered human secretory proteins, and an additional 400 human heterodimers, followed by a series of confirmatory screens, all performed on the Retrogenix cell microarray platform (Charles River). HTT was not a protein in the screening panel and was therefore spotted as an antigen in gelatin onto the screening slides as a positive control for the fixed format of the assay.

[0564] Live animal PET / CT scans to assess pharmacokinetics and brain penetration

[0565] To assess the pharmacokinetics and brain penetration of ATL_5895, a PET-labeled version of the antibody was prepared and PET in living animals as well as gamma counting in postmortem tissues were performed.

[0566] ATL-5895 was radiolabeled with zirconium-89 (89Zr) in a two-step procedure:

[0567] 1. ATL-5895 was first conjugated to the metal chelator deferoxamine (DfO) using the bifunctional chelator p-SCN-Bn-DfO. DfO-ATL-5895 was then purified by size exclusion chromatography (SEC).

[0568] 2. DfO-ATL-5895 was then radiolabeled with 89Zr at room temperature and the final product 89Zr-DfO-ATL-5895 was purified by SEC. After this, a 20 μL aliquot of 89Zr-DfO-ATL-5895 was injected onto a size exclusion HPLC system to allow assessment of radiochemical purity and DfO-ATL5895 concentration.

[0569] Transgenic female R6 / 1 mice (Jackson Laboratory Stock No: 006471) and age-matched C57BL / 6J controls at 11 to 12 weeks of age and 14 to 15 weeks of age were used. Mice were dosed with 100ul of 89Zr-Df-ATL5895, 1.5±0.3MBq, 1.15mg / kg. PET / CT scans were performed under anesthesia (1.5% to 2.5% isoflurane), and static PET images were acquired using Molecubes β-CUBE at 1, 24, 48, 72, and 168 hours after administration. After each PET scan, a CT scan was performed using Molecubes X-Cube. Image analysis was performed using PMOD software.

[0570] Blood samples were collected at 0, 1, 6, 12, 24, 48, 72, and 168 hours after administration by capillary tail method (20 μl), and 89Zr-Df-ATL5895 levels were assessed by γ counting. γ counting of isolated tissues was performed 168 hours after administration. A Perkin Elmer Wallac Wizard γ counter was used to obtain isolated organ biodistribution data.

[0571] Manufacturability—ATL5895

[0572] Thermal stability studies. ATLX_1095 (ATL_5895), expressed from CHO cells and purified in one stage, was subjected to temperatures of -80°C, +4°C, +21°C and +40°C at 5 mg / mL in 20 mM histidine-acetate, 150 mM NaCl, pH 5.5 for 4 weeks.

[0573] 10x Freeze-Thaw Cycle Study. ATLX_1095 (ATL_5895) at 5 mg / mL in 20 mM histidine-acetate, 150 mM NaCl, pH 5.5 was frozen at -80°C and thawed at room temperature (21°C) for a minimum of 30 minutes for 10 cycles over a 24 hour period.

[0574] Protein thermal shift and light scattering. Protein thermal shift measurements were performed in triplicate on Uncle (Unchained labs) on 5 mg / mL of unstressed ATLX_1095 (ATL_5895) in 20 mM histidine-acetate, 150 mM NaCl, pH 5.5. 8.8 μL of sample was loaded into three wells of Uni (Unchained labs—a proprietary strip of 16 9 μL quartz cuvettes placed in a blue metal frame with a silicone seal). The laser settings were set to achieve an initial fluorescence in the range of 300 to 350 nm of 10,000 to 50,000 counts. The antibody was ramped from 25°C to 95°C at a rate of 0.5°C / min and excited at 266 nm while monitoring fluorescence emission and SLS. Melting temperature (Tm1 / Tm2) and aggregation temperature (Tag (Taggregation) / Tonset (Tonset)) were analyzed using Uncle analysis software v6 (Unchained Labs). Tm measurements were calculated from the barycentric mean (BCM) of the fluorescence intensity curve from 300 to 430 nm, while Tagg and Tonset were calculated from the intensity of light scattered at 266 nm.

[0575] CE-SDS. CE-SDS analysis was performed on a Maurice (ProteinSimple, Bio-Techne). Samples were diluted to approximately 1 mg / mL and a volume of 50 μL with Protein Simple 1× sample buffer. To obtain the reduced sample, 2.5 μL of 14.2 M 2-mercaptoethanol was added. The sample solution was then transferred to a 96-well plate and centrifuged at 1000×g for 10 minutes before being placed in a Maurice. Injections were performed at 4600V for 20 seconds, and the reduced sample was separated at 5750V for 25 minutes. The results were analyzed using Compassfor iCE software (Bio-Techne) and Chromeleon software (Thermo).

[0576] cIEF. cIEF analysis was performed on a Maurice (ProteinSimple, Bio-Techne). A 5 mg / mL sample of ATLX_1095 (ATL_5895) in 20 mM histidine-acetate, 150 mM NaCl, pH 5.5 was diluted to approximately 1 mg / mL with ultrapure water. The antibody sample was added to a master mix containing 0.35% methylcellulose, 4% pharmalyte 3-10, 10 mM arginine, 0.01% pH 4.09 pI marker, and 0.01% 9.99 pI marker to a final antibody concentration of 0.15 to 0.25 mg / mL. Sample separation was performed at 1500 V for 1 minute, followed by x minutes at 3000 V. The results were analyzed using Compass foriCE software (Bio-Techne) and Chromeleon software (Thermo).

[0577] SEC-HPLC. Antibody samples were diluted to approximately 1 mg / ml in 20 mM histidine acetate, 150 mM NaCl pH 5.5 and filtered through a 0.22 μm filter. Approximately 25 μ g of the sample was loaded onto a Zorbax GF-250SEC-HPLC column (Agilent) or a TSKgel G3000SWxl column (TOSOH Bioscience) on a Vanquish Flex (Thermo) by injecting 25 μ L of 1 mg / mL sample. At 25 ° C, the sample was subjected to isocratic elution using 20 mM sodium phosphate, 300 mM sodium sulfate, and 100 mM arginine at a flow rate of 0.75 mL / minute, with each sample being eluted for 25 minutes (Zorbax column) or 40 minutes (TSKgel column). Chromeleon software (Thermo) was used to integrate the chromatogram to determine monomer purity.

[0578] HTT sandwich ELISA. In order to evaluate the binding efficacy of stressed antibody samples to HTT protein, the binding of antibodies to HTT exon 1 containing 48Q repeats (for antigen sequences, see Table 1) was evaluated in a sandwich ELISA format. Anti-HTT capture antibody (MerckMillipore; #MABN2427) was diluted to 4.17 μg / mL in 1×ELISA coating buffer (Biolegend; #421701). 50 μL was added to each well of a 96-well plate and placed at 4°C overnight. The next day, the plate was washed with PBS / 0.1% Tween. The plate was then blocked with 50 μL / well of blocking buffer (1% BSA / PBS). The plate was washed with PBS / 0.1% Tween. 50 μl of diluted antigen (HTT exon 1 48Q GST) and lysozyme (negative antigen control) were added to the plate at 0.04 μg / ml. The plate was incubated on a plate shaker (300 to 400 rpm) at RT for 1 hour. The plate was washed with PBS / 0.1% Tween. 8-point 3-fold serial dilutions of the test antibody and isotype control antibody (starting at 60 μg / mL) were added to the plate. The plate was incubated on a plate shaker (300 to 400 rpm) at RT for 1 hour. The plate was washed with PBS / 0.1% Tween. 50 μl of anti-human IgG HRP (80 ng / mL; Jackson ImmunoResearch, #109-035-097) was added to each well for 10 minutes. The plate was washed with PBS / 0.1% Tween, 50 μL of TMB solution (Lifetechnology, #002023) was added and the plate was incubated in the dark at room temperature for 6 to 9 minutes. 50 μL of stop solution (0.5 M sulfuric acid) was added to the plate (Fisher chemical, #12933634). Absorbance was read at 450 nm on a CLARIO Star. Data analysis was performed using GraphPad Prism 10 software (10.1.0.316) and EC50 (half maximal concentration) values were calculated using a nonlinear four-parameter curve fit without restraints.

[0579] ATL_5895 is produced for solubility assessment. Recombinant antibody ATL_5895 is transiently expressed by ExpiCHO-S cells using Expifectamine reagent (Thermo) according to the manufacturer's protocol. Transfection is cultured at 32°C for 13 days with the addition of feed, and then harvested by removing cells and mixing the supernatant with diatomaceous earth (Sartorius) and filtering through a 0.22 μm PES membrane. The harvested supernatant is purified using protein A chromatography and eluted with 50 mM sodium acetate pH 3.6. The eluted fractions are merged and the buffer is exchanged for 20 mM histidine acetate, 150 mM sodium chloride pH 5.5 and stored at 4°C, followed by long-term storage at -80°C.

[0580] Solubility assessment. Approximately 100 mL of 11.90 mg / mL ATL-5895 was concentrated to a maximum concentration of 89.96 mg / mL by tangential flow filtration (TFF) using a Minimate EVO tangential flow filtration system (Pall / Cytiva, PCode: OAPMPUNV) and a Minimate TFF Capsule 30K Omega membrane (Pall / Cytiva, PCode: OA030C12). 100 uL of sample was withdrawn from the sample reservoir at three time points. Sample concentration was determined by absorbance at 280 nm obtained by UV / Vis spectrophotometry on a Lunatic (Unchainedlabs).

[0581] Aggregation by SEC-HPLC assessment sample.Antibody sample is diluted to about 1mg / ml in 20mM histidine acetate, 150mM NaClpH5.5 and filtered by 0.22 μm filter.By the 1mg / mL sample of injecting 25 μ L, the sample of about 25 μ g is loaded on the TSKgelG3000SWxl post (TOSOH Bioscience) on Vanquish Flex (Thermo).At 25 ℃, with the flow of 0.75mL / minute, sample is carried out isocratic elution with 20mM sodium phosphate, 300mM sodium sulfate and 100mM arginine, and each sample carries out 25 minutes (Zorbax post) or 40 minutes (TSKgel post).Use Chromeleon software (Thermo) to integrate chromatogram to determine monomer purity.

[0582] Mouse HTT ELISA

[0583] To evaluate the binding potency of the antibodies to mouse HTT protein, the binding of ATL5895, ATL6376, and ATL6377 to mouse HTT and human lysozyme (as a negative control antigen) was evaluated in a direct ELISA format. Mouse HTT antigen (SEQ ID NO: 177) or lysozyme was directly absorbed into the ELISA plate at 3ug / ml (50ul per well) and incubated overnight at 4°C. The plate was washed with PBS. The plate was blocked with 200ul / well of blocking solution (1% BSA w / v in PBS) for 1 hour at room temperature. After this, the blocking solution was removed and the antibody to be evaluated was diluted in a dilution series (1uM to 0.05nM) in blocking solution (1% BSA w / v in PBS) and applied to the plate. The plate was incubated at room temperature for 1 hour. The plate was washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch; #109-035-097) was added to the plate and incubated at room temperature for 1 hour to detect antibody binding. The plate was washed with PBS / 0.1% Tween and TMB solution (Life Technology; #002023) was added. The plate was incubated at room temperature for 5 minutes, and then stop solution (0.5M sulfuric acid) was added. The absorbance at 450nm was read on a Molecular Devices FilterMax F5 plate reader. Analyzed using a nonlinear curve fitting algorithm on GraphPad prism, and EC50 was calculated.

[0584] Example 1 - Convergence analysis of AD cohorts to identify VH sequences associated with restoration

[0585] Convergent sequence clusters derived from the antibody repertoire of an individual's restorative group can be used to identify disease-specific antibody sequences. In the case of neurodegeneration, restorativeness can be defined as long-term symptom-free status despite a strong predisposition to the disease.

[0586] Cognitive scores and biomarkers were used to identify candidate protective antibodies from a recovering subgroup of patients at risk within a cohort of patients at risk of dementia (in collaboration with the European Prevention of Alzheimer's Dementia (EPAD)). Figure 1AThe workflow used in this example to identify convergent VH sequences from an AD dataset is shown. Restorativeness is defined as a significant reduction in β-amyloid in the cerebrospinal fluid (CSF) of patients at risk for Alzheimer's disease (AD) compared to healthy controls, which is closely associated with increased β-amyloid deposition in the brain. This subgroup of patients also had low levels of pTau in the CSF, indicating significantly less neuronal damage than AD progressors, and exhibited sustained normal cognitive function compared to age-matched individuals who were progressing with unfavorable β-amyloid and p-Tau CSF biomarkers.

[0587] The present inventors identified a cluster of related antibody heavy chains that were convergent in recovered individuals but absent in control individuals ( Figure 1B ).exist Figure 1B Above, the sequence labeled "known HTT binder" is the sequence labeled ATL_0005059 herein, also known as NI-302.8F1, and described in US11,401,325B2. It is important to note that the antibodies described herein were identified by a process independent of this previous antibody. In fact, the antibodies of the present invention were identified as related antibody heavy chains that are convergent in individuals with restorative properties to neurodegenerative diseases. These antibodies were identified as binding to HTT and were therefore compared with known antibodies for context. In other words, the antibodies currently described were not developed by modifying existing antibodies. Instead, the antibodies currently described were discovered by analyzing naturally occurring protective (and therefore potentially therapeutically effective) antibodies and their optimization, wherein the identification was entirely target- and sequence-independent, and the optimization was entirely independent of prior art antibodies, but rather was further improved upon already excellent antibodies derived from naturally occurring sequences. These antibodies exhibit features of immune activation, supporting their role in restorative properties. To deconvolute the targets of this VH cluster, we compared them to a database of antibodies with known binding specificities curated from the literature. One VH in this cluster had a CDR3 amino acid sequence identical to a known binder of huntingtin, a protein encoded by the Htt gene and closely associated with Huntington's disease. This finding suggested that the convergent VHs in this cluster might also bind to the HTT protein.

[0588] Two of these VHs (ATL5060 and ATL5061—see Tables 1 and Figure 1B ) were expressed as antibodies using the light chain (VL) from a known binder. Both antibodies were confirmed to bind to HTT by ELISA.

[0589] The discovery of antibodies against HTT in patients with recovered AD 'risk' can be considered unexpected. Aggregated and mutated HTT (mHTT) proteins have been reported as the main cause of Huntington's disease, however, the presence of mutated or aggregated proteins has been associated with other neurodegenerative diseases. HTT aggregates accumulate in the cytoplasm of dystrophic neurons in the AD brain and in microglia (Singhrao, S et al. Huntingtin Protein Colocalizes with Lesions of Neurodegenerative Diseases: An Investigation in Huntington's, Alzheimer's, and Pick's Diseases. Exp Neurol 150, 213 (1998)) and in another AD study, they accumulate in neurons in the prefrontal cortex and hippocampus (Axenhus, M, et al. Huntingtin Levels are Elevated in Hippocampal Post-Mortem Samples of Alzheimer's Disease Brain. Curr Alzheimer Res 17, 858 (2020)). The accumulation of HTT is associated with the formation of tau fibrils and tangles in both HD and AD (Masnata, M, et al. Targeting Tau to Treat Clinical Features of Huntington's Disease. Front Neurol 11, 580732 (2020)). In addition, a small proportion of patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) have also been reported to have CAG codon expansions (CAG>40) in Htt (the ratio is 4.4 times higher than that in healthy individuals) (Dewan, R. et al. Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis. Neuron 109, 448 (2021)).Additionally, it has been shown that mHTT polyglutamine expression enhances the seeding properties of aggregated TDP-43 in cell models (Coudert, L. et al. Phosphorylated and aggregated TDP-43 with seeding properties are induced upon mutant Huntingtin (mHtt) polyglutamine expression in human cellular models. Cell Mol Life Sci 76: 2615 (2019)). Therefore, HTT dysfunction may play an etiological role in a wider group of neurodegenerative diseases than previously thought, and at an earlier stage of pathology than changes mediated by Tau, β-amyloid, or TDP-43.

[0590] In addition to the two individuals from the EPAD cohort, the homologue was also found in another individual with AD from another cohort, as well as in two individuals who were genetically susceptible to FTD but showed resilience to FTD (data not shown). This further supports the role of mHTT in multiple neurodegenerative disorders.

[0591] Example 2—Phage display for optimal VJ / VL pairings

[0592] Based on the discovery of antibody ATL_5060, combinatorial phage display was used to identify the best VL partners for VH. The VH sequence from ATL_5060 was combined with >1 million potential VL partners in the phage display library and then selected on HTT protein to enrich for functional binders. This process was performed in Figure 2A and B are shown.

[0593] Phage selection generated seven unique scFv sequences (labeled ATL_0005331 to 5337), and mHTT phage ELISA indicated two strong binders (ATL_5331; ATL_5335) and one moderate binder (ATL_5334) ( Figure 3A After phage display enrichment, the resulting human IgG1 antibodies containing the selected VL sequences were prepared and tested for binding to HTT protein by ELISA ( Figure 3B ). Figure 3A and 3BBoth assays shown are indirect ELISAs that measure binding to mHTT coated on a plate. However, the test samples differed between A and B. In A, the test samples were phage displaying antibody fragments corresponding to the selected antibody sequence. In B, the test sample was an antibody sequence formatted as human IgG. The experiment shown in A was a screening experiment in which a higher concentration of mHTT was used to ensure detection of binding levels. The experiment shown in B was a determination of binding equilibrium in which the concentration of mHTT used gave increased sensitivity. The minimal differences in absorbance observed in A are likely due to the variability of the phage in each test sample, including different display levels of the antibody fragments and different concentrations of phage in the test samples applied to the experiments, which resulted in variable concentrations of the antibody fragments in the test samples. In contrast, in B, there was little variability in the amount of antibody used in the test sample, as this can be easily measured based on protein concentration.

[0594] ATL_5331, ATL_5334, and ATL_5335 were selected for further optimization.

[0595] Figure 4A Convergent VH sequences identified in an AD-recovered individual (ATL_0005042) are shown. Figure 4B Functionally paired VLs (ATL_0005331-5335) from phage display selections on HTT exon 1 and sequence analysis are shown aligned with CA_0000274 VL (also referred to herein as ATL_0005059 or NI-302.8F1 and described in US 1 1 ,401 ,325 B2).

[0596] Example 3—Epitope Mapping

[0597] To evaluate the epitopes of antibodies against the HTT protein (ATL_5331; ATL_5566 (=ATL_5334 with free cysteines removed) and ATL_5335), the antibodies were evaluated against peptide arrays constructed from peptide fragments corresponding to the sequence of human, cynomolgus monkey (cyno, 2 different reference sequences that could be cynomolgus monkey HTT protein sequences) and mouse HTT exon 1.

[0598] The peptide array consisted of 15-mer linear peptides with 14 amino acid overlap on HTT exon 1 (see Figure 5 ), allowing high-resolution epitope mapping. For HTT exon 1, species cross-reactivity was tested in parallel using two cynomolgus monkey isoforms and one mouse isoform.

[0599] Table 2 shows the sequences of the peptides used to generate the array.

[0600]

[0601] Table 2 - Reference sequences used to generate peptides.

[0602] The resulting huntingtin peptide microarray contained HTT peptides printed in duplicate and framed with additional HA (YPYDVPDYAG, 48 spots) and polio (KEVPALTAVETGAT, 48 spots) control peptides. The huntingtin peptide microarray was incubated with antibody samples at concentrations of 1 μg / ml and 10 μg / ml, then stained with secondary antibody (0.2 μg / ml goat anti-human IgG (H+L) DyLight680) and control antibody (0.2 μg / ml mouse monoclonal anti-HA (12CA5) DyLight800) and read out using an Innopsys InnoScan 710-IR microarray scanner. The analyzer performs spot intensity quantification and peptide annotation. Briefly, spot intensity quantification and peptide annotation are based on 16-bit grayscale tiff files. The analyzer performs microarray image analysis. The fluorescence intensity of each spot is decomposed into raw signal, foreground signal and background signal, and the median foreground intensity (called "corrected intensity") and the spot-to-spot deviation of the duplicate spots are calculated. The maximum inter-spot deviation is allowed to be 40%, otherwise the corresponding intensity value is reset to zero. The average spot intensity plot obtained by measuring the antigen sequence from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein for human antibody samples is used to visualize the overall spot intensity. The intensity plot is associated with the peptide and intensity map and combined with visual inspection of the microarray scan to identify the epitope of the antibody sample.

[0603] Figures 6 to 8 Shown are three selected monoclonal antibodies (mAbs) and their binding strength to the HTT exon 1 region. All mAbs showed similar binding profiles across species, and binding was observed primarily at two different sites in HTT exon 1. The alignment revealed a common motif (in Figures 6 to 8 (Indicated in bold letters in the lower panel) A binding consensus motif shared between the binding sites on exon 1 was identified as [P / Q]Q[P / Q]QPPPPPPPPP (SEQ ID NO: 113).

[0604] Table 3 is a summary of peptides for which binding was detected for at least one antibody concentration.

[0605]

[0606]

[0607]

[0608]

[0609] Table 3 - Summary of peptides detected binding to HTT exon 1 peptides. Sp = species, H = human, M = mouse, C1 = cyno_1, C2 = cyno_2. Data = normalized fluorescence intensity.

[0610] In summary, epitope mapping revealed that the three selected antibodies had complex binding sites in HTT exon 1 and preferentially bound to peptides with multiple C-terminal prolines.

[0611] Example 4—Stability Study

[0612] To evaluate the stability characteristics of HTT exon-1 mAbs, a 3-week accelerated stability study was performed on the three selected mAbs and their fully germlined equivalents: ATL_5331; ATL_5334; ATL_5335; ATL_5555 (fully germlined ATL_5331); ATL_5556 (fully germlined ATL_5334); and ATL_5557 (fully germlined ATL_5335). The workflow for the stability study is described in Figure 9 Shown in.

[0613] Antibodies were stored at -80°C, 4°C, room temperature, or 40°C for three weeks, and samples were subjected to five freeze-thaw cycles from -80°C to room temperature. Quality control analyses were then performed, including assessment of purity, aggregation, degradation, charge variants, and thermal stability.

[0614] Protein aggregation during antibody storage must be kept to a minimum as it can induce immunogenic responses. Size exclusion chromatography (SEC)-HPLC was used to evaluate the purity and aggregation of antibodies in a 3-week stability study and after 5× freeze-thaw cycles.

[0615] Figure 10 shows the absence of soluble aggregate formation (SEC-HPLC) after 3 weeks incubation at -80°C, 4°C, room temperature (21°C) and 40°C. Table 4 further supports the excellent stability of the six antibodies tested and shows low levels of high molecular weight species (HMWS) and low levels of low molecular weight species (LMWS), indicating that the antibodies have a low tendency to aggregate and degrade up to 40°C, which is further supported by the high percentage of monomers >95% in each sample throughout the study.

[0616]

[0617] Table 4 - Results from SEC-HPLC. RT = room temperature 21°C; FT = freeze-thaw; HMWS = high molecular weight species; LWMS = low molecular weight species.

[0618] After a low pH hold step (at pH 3.5) for up to 120 minutes, antibodies ATL_5331 or ATL_5335 did not form detectable soluble aggregates (SEC-HPLC) ( Figure 11 and Table 5 ).

[0619]

[0620] Table 5 - Low pH hold SEC-HPLC results

[0621] Capillary isoelectric focusing (cIEF) was used to assess the charge heterogeneity of the antibodies. The isoelectric points (pI) of all six antibodies were determined using cIEF before subjecting the antibodies to temperature stress or freeze-thaw cycles (see Figure 12A and Table 6). The pI values of the antibodies were in the typical range of 7.5 to 9, suitable for downstream processing.

[0622] Antibody Main Peak pl Main peak area (%) ATL_0005331 8.68 68.5 ATL_0005334 8.87 61.9 ATL_0005335 8.52 62.6 ATL_0005555 8.93 62.9 ATL_0005556 9.08 63.4 ATL_0005557 8.89 63.0

[0623] Table 6 - Main peak pi and main peak area (%) for the six designated antibodies shown in Figure 12 before stability studies (T=0).

[0624] After three weeks of incubation at 40°C, an increase in acidic charge variants was observed under the highest temperature stress condition, but not at any other tested temperature (Figure 12B) or after 5× freeze-thaw cycles (Figure 12C). In summary, the biophysical properties of the antibody did not undergo significant changes in the accelerated temperature stress studies conducted at 5 mg / ml in non-optimized formulation buffer.

[0625] Example 5—In vivo PK profile

[0626] To determine the serum PK of the lead antibody, an in vivo PK experiment was performed with ATL_0005335 (the parent antibody of ATL_0005567). Antibody levels were assessed by ELISA of serum samples collected 1, 4, 8, 24, 72, and 144 hours after administration. ATL_0005335 showed linear PK and did not show altered clearance compared to the non-binding IgG1 antibody in wild-type animals. This suggests that the antibody does not unexpectedly bind to molecules outside the CNS, which will have implications for pharmacokinetics and potential safety.

[0627] Example 6—Optimization of lead antibodies

[0628] To identify the best candidates for preclinical testing and ultimately for testing in the clinic, e.g. Figure 14 As shown in , the antibodies were subjected to multiple rounds of triaging. In order to identify the lead antibodies with the best development characteristics, a first set of antibodies was designed and consisted of variants of ATL_5331; 5334; and 5335 containing germline intermediate mutations and liability removal mutations. Reverting to the germline framework sequence reduces the potential immunogenicity of the antibody, while mutating the sequence motifs to remove potential liability can help minimize future manufacturing and pharmacokinetic issues caused by amino acid modifications.

[0629] Tables 7 and 8 show the VH and VL sequences of the parent antibodies, their germline equivalents, and equivalents with additional development mutations.

[0630]

[0631]

[0632] Table 7 - VH sequences of test antibodies and prior art antibody ATL_5059.

[0633]

[0634]

[0635]

[0636] Table 8 - VL sequences of test antibodies and prior art antibody ATL_5059

[0637] A total of 69 mAbs (monoclonal antibodies) were screened based primarily on binding to HTT exon 1 in ELISA and thermal stability analysis by using SYPRO in a thermal shift assay. TM Orange measured Tm to determine (see Figure 15 ). For all variants, there was no difference in thermal stability and had similar Tm (Tm1 (mean) = 62.4 ± 2.1 °C (%RSD)) ( Figure 15 ). Comparing the binding ELISA, all 5331 variants had high rankings, while the 5334 variants had variable rankings, and the 5335 variants had low rankings.

[0638] A second variant panel containing combinations of selected mutations was then designed, and the antibodies were further screened based on their binding to HTT exon 1 and thermal stability to select lead antibodies.

[0639] Lead antibodies were selected primarily based on affinity for HTT (using a 1-site binding ELISA) and binding potency as determined by sandwich HTT ELISA (ranked by: 48Q HTT Exo-1 EC50, E50 < 50 nM; then ranked by EC50 (25Q:48Q) and 48Q), and secondarily, based on germline mutations and removal of unfavorable mutations (removal of low-yielding mutations, stability markers, etc.; for example, G54A removes the aspartate isomerization motif).

[0640] Antibodies with lower EC50 values have higher binding potency to HTT exon 1 as determined by HTT sandwich ELISA, and therefore have higher affinity for HTT exon 1. First, antibodies were ranked based on their EC50 values for binding to 48Q HTT exon-1. Antibodies with EC50s below 50 nm were screened and further ranked based on EC50 and 25Q:48Q ratio. The higher the ratio of 25Q:48Q binding, the more potent the antibody is at 48Q HTT over 25Q HTT (i.e., the lower the ec50 of 48Q vs. the ec50 of 25Q, the better). Other factors considered in selecting lead antibodies are the number of germline mutations and adverse factor removal mutations, as these antibodies pose fewer safety concerns.

[0641] Finally, three lead antibodies ATL_5901 (5334 variant, ranked 1); ATL_5895 (5331 variant, ranked 1); and ATL_5567 (5335 variant, ranked 3) were selected.

[0642] ATL_5901 and ATL_5895 had similarly low EC50 levels (see Figure 16 and Tables 9 to 10) and contained developmental and germline mutations. ATL_5567 was the best performing 5335-derived variant.

[0643]

[0644] Table 9 - EC50 (nM) of antibodies against HTT exon 125Q

[0645]

[0646] Table 10 - EC50 (nM) of antibodies against HTT exon 148Q

[0647] Ratio 25Q:48Q ATL_0005895 1.785991 ATL_0005901 2.052968 ATL_0005567 1.183835 ATL_0005059 / NI-302.8F1 1.119093

[0648] Table 11 - EC50 ratios of 25Q:48Q HTT exon 1

[0649] Compared to the reference antibody NI-302.8F1 described in US11,401,325B2, all three lead antibodies consistently showed higher binding potency (up to 9-fold) and higher 25Q:48Q ratios (Table 11) for HTT exon 125Q (Table 9); HTT exon 148Q (Table 10).

[0650] These data therefore demonstrate that the three lead antibodies, ATL_5901, ATL_5895, and ATL_5667, have improved binding properties compared to prior art antibodies. The lead antibodies were also optimized for germline and sequence-removal mutations to potentially reduce immunogenicity and increase shelf life, stability, and manufacturing suitability.

[0651] The manufacturing suitability of ATL_5895 was further confirmed by verifying that no aggregation was observed (by SEC-HPLC) after 4 weeks of incubation at 40°C (vs. -80°C) as described above, and no aggregation was observed (by SEC-HPLC) after 10 freeze-thaw cycles as described above. ATL_5895 also has a pI in the appropriate range for downstream processing (i.e., 7.5E-9).

[0652] Example 7—Binding to Disease-Related HTT

[0653] Huntington's disease is caused by a pathological expansion of a cytosine-adenine-guanine (CAG) triplet repeat in the huntingtin (HTT) gene, which results in the production of mutant HTT proteins. As shown in Example 6, ATL_5331; 5334; 5335; 5895; 5901; and 5567 were all able to bind to 48Q HTT exon 1 (i.e., mutated HTT) in an ELISA, indicating that these antibodies can bind to pathological length forms of HTT. In order to determine whether the antibody can bind to high molecular weight HTT (meaning HTT that is more in a pathological aggregate-like state), immunoprecipitation experiments were performed using ATL_0005335 (the parent antibody of ATL_0005567).

[0654] To confirm that the antibodies of the invention can bind to the disease-associated HTT, the binding of ATL_5335 to the U-2OS cell line, which expresses 110 CAG repeats and produces a high molecular weight species of HTT (HTT), was tested by Western blotting after immunoprecipitation with ATL_5335. 110 ), similar to the mutant HTT protein. Figure 17A As shown in , ATL_5335 can immunoprecipitate HTT with a molecular weight > 180 kDa (HTT 110 ).

[0655] R6 / 2 transgenic mice express the 5' end of the human HTT gene, including exon 1 with approximately 120 CAG repeats, and exhibit neurological phenotypes similar to those characteristic of HD in humans, including the development of pathological HTT aggregates. Figure 17B It was shown that ATL_0005895, ATL_0005901 and ATL_0005567 were able to immunoprecipitate high molecular weight HTT (in this case >250 kDa, which is the highest molecular weight protein marker used in this gel ladder) from R6 / 2 mouse brain homogenate. These data therefore indicate that the antibodies of the invention are able to bind aggregated HTT protein.

[0656] To confirm that ATL_0005895 can bind to disease-associated HTT in human brain, brain homogenates were prepared from superior temporal gyrus tissue from postmortem brains of Huntington's disease patients. The material was incubated with beads coupled to an isotype control antibody or ATLX1095 and subsequently analyzed by Western blotting. High molecular weight HTT material was successfully immunoprecipitated by ATL_0005895 but not by the isotype control antibody ( Figure 17C and 17D ). In particular, Figure 17CATL_0005895 was shown to be able to immunoprecipitate high molecular weight HTT from human brain homogenates derived from Huntington's disease patients, as shown in Figure 17D These data demonstrate that the antibodies of the present invention are able to bind to highly disease-associated aggregated HTT protein from human brain tissue.

[0657] Example 8—Binding of anti-HTT antibodies to seeding-competent HTT species

[0658] In many neurodegenerative diseases, including HD, which is characterized by pathological aggregation of toxic mHTT species, self-propagating protein aggregates drive pathogenesis. Therefore, it was of interest to test the ability of ATL_5895 and ATL_5901 to bind to these seeding-competent HTT species and inhibit pathological HTT aggregation.

[0659] To test the anti-seeding potential of anti-HTT antibodies ATL_5895 and ATL_5901, the aggregation rate of fluorescence resonance energy transfer (FRET)-labeled recombinant HTT was assessed by FRET-based mHTT aggregate seeding (FRASE) assay as described in Ast, Anne et al. "mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease." Molecular cell vol. 71, 5 (2018): 675-688.

[0660] FIG. 18 shows the use of recombinant HTT seeds ( Figure 18A ) and brain homogenates from R6 / 2 mice ( Figure 18B ) were used to perform FRASE assays. Immunodepletion of pathological HTT or “seeds” with antibodies ATL_0005895 and ATL_0005901 reduced the in vitro aggregation rate of seeding-competent HTT species. Notably, the mouse monoclonal IgG2A antibody MW8 (Millipore; MABN2529) against human HTT bound to aggregated HTT and also achieved this effect to some extent, whereas MW1 (Millipore; MABN2427), which binds to the PolyQ region of HTT, did not achieve this (see Figure 18A and B).

[0661] Together, these results demonstrate that the antibodies described herein are able to reduce the rate of self-propagation of toxic HTT species, suggesting that these antibodies may be able to slow disease progression by reducing HTT seeding.

[0662] Example 9—Phagocytosis Assay

[0663] As illustrated above in Examples 7 and 8, HD is characterized by the aggregation of mHTT species that have the ability to self-propagate / seed, thereby driving HD pathogenesis. An important immune defense mechanism in the central nervous system is the phagocytic clearance of neurotoxic proteins, such as these mHTT species, by microglia.

[0664] To investigate whether binding of the anti-HTT antibody ATL_5895 to exon 1 of HTT improves phagocytic clearance of HTT by microglia, an in vitro assay using induced pluripotent stem cell (iPSC)-derived microglia was used to monitor phagocytic activity in culture (see the Materials and Methods section above).

[0665] Figure 19 Shown are the results of the assay of anti-HTT antibody ATL_5895 versus isotype control antibody ATL5338 (which binds fluorescein). Figure 19 It is shown that binding of ATL_5895 increased the uptake of 48Q HTT-coated beads in iPSC microglia in a dose-dependent manner compared to beads treated with an isotype control.

[0666] The results of this experiment indicate that the antibodies described herein increase the phagocytic clearance of toxic mHTT species.

[0667] Example 10—In vivo PK study

[0668] The pharmacokinetic properties of selected anti-HTT antibodies were tested in vivo to assess circulating levels and CNS penetration of the antibodies.

[0669] Antibody levels were assessed by ELISA on serum samples collected 1, 4, 8, 24, 72, and 144 hours after administration, and on CSF samples collected 4 and 144 hours after administration.

[0670] Figure 20 shows the results of the PK study. When administered at a concentration of 10 mg / kg, ATL_5901 and ATL_5567 exhibited linear serum PK profiles ( Figure 20A ). Figure 20B Serum concentrations of ATL_5895 following treatment with 1, 10, or 60 mg / kg ATL_5895 are shown and all of these concentrations produced linear serum PK profiles. Importantly, ATL_5895 also showed evidence of CNS penetration ( Figure 20CTypical penetration of IgG1 in the CNS is 0.1% to 0.3%. This data suggests that CNS exposure exceeding the EC50 of the antibody is achievable.

[0671] Proof-of-concept pharmacology studies in the R6 / 1 mouse model are described in Example 15.

[0672] Example 11—Affinity maturation of ATL_5895

[0673] As demonstrated in the above examples, ATL_5895 binds to disease-associated HTT, inhibits seeding, and increases phagocytic clearance of pathological HTT. Therefore, the present inventors investigated whether there was room for affinity maturation of the ATL_5895 antibody in order to identify additional high-affinity HTT-binding antibodies.

[0674] Affinity maturation of ATL_5895 was performed by phage display, wherein the phage library contained one or more mutants of the following amino acids: YCIPPPYYYYYGLDV sequence ("extended CDR3H") in the VH of ATL_5895; GSYAGTANV sequence (CDR3L) in the VL of ATL_5895. "Extended CDR" refers to a region comprising CDR3H and a position outside CDR3H, in this case four amino acids. In this case, 103 is one of the positions selected for "hard randomization" (described under "phage display" in the "Materials and Methods" section above). Residues close to but outside the CDR are mutated to introduce more subtle changes in the function of the antibody. Three rounds of phage selection were performed for human HTT exon 1 48Q or biotinylated GYSLPQPQPPPPPPP peptide. The extended CDR3H and CDR3L regions of the antibodies identified by affinity maturation by phage display are shown in Table 12. The full VH and VL sequences of these antibodies are provided in Table 1.

[0675]

[0676] Table 12 - Sequences of extended CDR3H and CDR3L of antibodies identified by phage display. HCDR3 is located between positions 95 and 102 according to Kabat numbering. The extended CDR3H comprises positions 91 to 102. The extended CDR3H shown above comprises positions 91 to 102.

[0677] Figures 21 and 22 show the results of indirect ELISA and sandwich ELISA, respectively, for binding of the newly identified antibodies to exon 1 48Q HTT. Table 13 shows the EC50 values derived from the sandwich ELISA results in Figure 22.

[0678]

[0679] Table 13 - EC50 values derived from sandwich ELISA.

[0680] The above data demonstrate that additional antibodies identified by phage display are able to bind to exon 148Q HTT with high affinity.

[0681] Antibodies ATL_6205, ATL_6202, ATL_6194, and ATL_6203 were used as the basis for further optimization in Example 17. Antibody ATL_6195 was also selected for further study because homologous sequences were found in HD recovering patients.

[0682] Example 12—HD patients produce HTT-binding antibodies

[0683] To identify additional anti-HTT binding antibodies, the present inventors investigated the B cell repertoire of brain samples from patients diagnosed with HD by sequencing to identify antibodies homologous to the antibodies described herein. The brain samples were obtained from the European Network of Brain Banking (ENBB). The identification of anti-HTT binding antibodies in HD patients further supports the disease relevance of the antibodies of the present disclosure.

[0684] Two antibodies with homology to ATL_5895 were identified in the same ENBB patient and paired with the VLs of ATL5895 and ATL5901 to generate four new antibodies. The two homologous antibodies identified from the ENBB patient showed differences in the FW region compared to ATL5895 and ATL5901. Therefore, four additional variants were generated that contained only the HCDR3s of the two homologous antibodies, with the remainder of the antibody being ATL5895. Table 14 contains details about the VH and VL pairings and CDR3 usage of the newly generated antibodies.

[0685] Table 16 shows the VH CDR sequences for each of these antibody variants, as well as the VH CDR sequences for ATL5895 and ATL5901. The full VH and VL sequences for these antibodies are provided in Table 1.

[0686] Antibody VH VL ATL6183 ENBB homologous antibody VH1 CDR3; ATL5895VH ATL5895VL ATL6184 ENBBB homologous antibody VH1 CDR3; and ATL5901VH ATL5901VL ATL6185 ENBB homologous antibody VH2 CDR3 and ATL5895VH ATL5895VL ATL6186 ENBB homologous antibody VH2 CDR3 and ATL5901VH ATL5901VL

[0687] Table 14 - VH and VL pairings of novel antibody variants

[0688] Figure 23 shows the results of sandwich ELISA of newly identified antibodies binding to exon 148Q HTT. All antibodies showed binding to HTT, while control antibody ATL_5338 (isotype control, conjugated to fluorescein) did not bind to HTT. Table 15 shows the EC50 values derived from the sandwich ELISA results shown in Figure 23.

[0689]

[0690] Table 15 - EC50 values derived from sandwich ELISA for the indicated antibodies.

[0691] Taken together, these data confirm that the antibodies of the invention are capable of binding disease-associated HTT.

[0692] Antibody VH CDR1 VH CR2 VH CDR3 ATL6183 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYGLDV ATL6184 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYGLDV ATL6185 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYGLNV ATL6186 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYGLNV ATL5895 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYYGLDV ATL5901 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYYGLDV ATL5567 KAWMN RIKSGIDGGTTDYAAPVQG PPYYYYYGLDV

[0693] Table 16. VH CDRs of patient-derived antibodies.

[0694] Figure 24 shows the sequences of the FW regions and CDRs of the VH (Figure 24A) and VL (Figure 24B) of some antibodies of the present disclosure.

[0695] Example 13—In vitro selectivity assay

[0696] To investigate the selectivity of the antibodies, ATL_5895 and ATL_5567 were first screened for binding to fixed HEK293 cells overexpressing 6105 separate full-length human plasma membrane proteins, secreted and cell surface tethered human secretory proteins, and another 400 human heterodimers to identify library interactions. This library screen was followed by a series of confirmatory screens in which all library interactions were re-expressed in fixed and living cells and probed with each test antibody or control treatment to determine which interactions were reproducible and specific for each test antibody (see "Materials and Methods" section above). This was performed on both fixed and living cells. HTT was spotted as an antigen in gelatin onto a screening slide as a positive control (because HTT was not part of this particular screening panel).

[0697] Both ATL_5895 and ATL_5567 strongly bound to the HTT positive control (2 replicates per condition, 2 μg / mL antibody for ATL5895 and 5 μg / mL antibody for ATL5567 and isotype control ATL5338). Another poly Q protein, CACNA1A, was present but not detected by any of the antibodies. ATLX-1095 and ATL5567 did not show confirmed hits in this screening panel, demonstrating their selectivity. Rituximab was used as a positive control for CD20 and, as expected, was a hit for this antigen.

[0698] This data demonstrates that the ATL_5895 and ATL_5567 antibodies described herein specifically bind to HTT.

[0699] Example 14—Live Animal PET / CT Scans to Assess Pharmacokinetics and Brain Penetration

[0700] To assess the pharmacokinetics and brain penetration of ATL_5895, a PET-labeled version of the antibody was prepared and PET in living animals as well as gamma counting in postmortem tissues were performed (see Materials and Methods above).

[0701] Figure 25 shows the results of live animal PET experiments and gamma counting assays. For labeled antibodies in 11-12 and 14-15 week old cohorts of both C57BL / 6J and R6 / 1 mice, half-lives ranged from 8 to 9 days ( Figure 25A and 25B The mean brain:blood ratio for both groups was estimated to be 0.03±0.004 at 11 to 12 weeks and 0.04±0.01 at 14 to 15 weeks, as calculated by postmortem gamma counts ( Figure 25C and 25D As shown in Example 10, standard / classical PK experiments showed an exposure of approximately 5.5 nM in CSF ( Figure 20C ), suggesting that brain levels in this experiment may be underestimated. Live animal PET showed typical human IgG1 biodistribution of the labeled antibodies, with most of them detected in the blood ( Figure 25E and 25F )—although, as noted above, antibodies were also detected in the brain and CSF.

[0702] Example 15—In vivo PD assay

[0703] R6 / 1 is a transgenic mouse model of Huntington's disease that exhibits a progressive neurological phenotype that mimics many of the characteristics of Huntington's disease (Mangiariniet al; Cell; 1996) including the accumulation of aggregates over time (Hansson et al; EJN; 2001). These mice universally express a transgene that contains the 5' end of a mutant human huntingtin protein, which contains approximately 1 kb of 5'UTR sequence, exon 1 (carrying an expanded CAG repeat with 115 to 150 CAG repeats) and the first 262 bp of intron 1. To evaluate the effect of ATL_5895 (ATLX_1095) on the HTT aggregate load in R6 / 1 mice, mice were treated with vehicle or ATL_5895 for 12 weeks (starting at 5 weeks of age). Immunoassay (MSD) was used to assess the HTT aggregate load in the brains of these mice. Soluble mutHTT (2B7; MW1+) was assessed in plasma and an increase was observed, suggesting that target engagement and clearance of HTT is driven by antibody-HTT complexes in plasma.

[0704] Figure 26 shows the results of mesoscale discovery (MSD) assays for evaluating the effect of ATL_5895 (ATLX_1095) on HTT aggregate load in the striatum and cortex of R6 / 1 mice. As expected, in R6 / 1 mice, HTT aggregates increased over time in the striatum and cortex (4C9 / MW8+HTT; Figure 26A ), and at the same time, soluble mutant HTT decreased over time as assessed by MSD assay (2B7 / MW1). Treatment of R6 / 1 mice with ATL_5895 for 12 weeks resulted in a statistically significant reduction in HTT aggregates in the striatum and cortex (as detected by MW8 / 4C9+ antibody) ( Figure 26B There was no reduction in soluble HTT (as detected by 2B7 / MW1 antibody) or endogenous mouse HTT (as detected by 2B7 / D7F7 antibody) in the striatum and cortex (see Figures 2 and 3, respectively). Figure 26C and 26D Together, these results suggest that ATL_5895 (ATLX_1095) can selectively reduce HTT aggregates in the striatum and cortex of the R6 / 1 mouse model of Huntington's disease without affecting endogenous HTT levels.

[0705] Plasma neurofilament light (NEFL) levels were assessed, but no differences were observed between WT and R6 / 1 mice, thus there was no phenotype that the antibody was intended to rescue.

[0706] Example 16—Manufacturability

[0707] A stability assessment of the antibody ATLX-1095 (ATL_5895) was also performed, including a 4-week thermal stability study, a 10× freeze-thaw cycle study, a thermal stability assessment (Tm and Tagg), and a solubility study. The biophysical properties of the antibody were evaluated after exposure to different stress conditions, including (1) a 4-week thermal stability study in which the antibody was subjected to temperatures of -80°C, +4°C, +21°C, and +40°C; and (2) 10× freeze-thaw cycles. The stressed antibody samples were evaluated for purity, aggregation, degradation, and changes in charge heterogeneity and binding using SEC-HPLC, CE-SDS, cIEF, and ELISA compared to -80°C control conditions.

[0708] The melting temperature (Tm) and aggregation temperature (Tagg) of unstressed antibodies were assessed by DSF and SLS.

[0709] Antibody solubility was assessed by concentrating ATLX_1095 (ATL_5895) to 89.96 mg / mL using tangential flow filtration (TFF). Aggregation of samples was assessed by SEC-HPLC analysis after concentration and after 1 week at 21°C.

[0710] Protein aggregation during antibody storage must be kept to a minimum as it can induce immunogenic responses. Size exclusion chromatography (SEC)-HPLC was used to evaluate the purity and aggregation of antibodies in a 4-week stability study and after undergoing 10× freeze-thaw cycles. Figure 27A A to B show SEC-HPLC chromatograms of ATL_5895 after 4 weeks of thermal stability and 10× freeze-thaw cycles, respectively. After 4 weeks of incubation at -80°C, 4°C, room temperature (21°C), and 40°C, respectively, and after 10× freeze-thaw cycles, no increase in soluble aggregate formation was observed. The results in Table 17 show that monomer purity was consistently high (>95%).

[0711]

[0712] Table 17. Summary of SEC-HPLC ATL_5895-002 4-week stability study and 10× freeze-thaw study SEC-HPLC data showing monomer purity (%), high molecular weight (HMWS) species (%), and low molecular weight (LMWS) species (%).

[0713] Capillary isoelectric focusing (cIEF) was used to evaluate the charge heterogeneity of the antibody samples. cIEF was used to determine the isoelectric point (pI) of unstressed ATL_5895. The pI of ATL_5895 was 8.93, which is within the typical range of 7.5 to 9 for antibodies and is suitable for downstream processing (main peak area 71.84%). Changes in charge heterogeneity after temperature stress (-80°C, 4°C, room temperature (21°C) and 40°C for 4 weeks) and 10× freeze-thaw cycles were evaluated by cIEF. After incubation at 40°C for 4 weeks, an increase in acidic and basic charge variants and a decrease in the main peak were observed under the highest temperature stress condition, but no significant difference was observed at any other tested temperature ( Figure 27C ) or after 10× freeze-thaw cycles ( Figure 27D ) were not observed. These results are also summarized in Table 18, which shows the % basic and acidic species that were stable after most treatments (major isoform % approximately 70%).

[0714]

[0715] Table 18. Capillary isoelectric focusing results of ATL_5895 after temperature stress or 10× freeze-thaw cycles

[0716] Figure 27E and Table 19 show the results of CE-SDS performed on samples of antibodies subjected to the indicated treatments (reduction prior to CE-SDS).

[0717]

[0718] Table 19. Reduced CE-SDS light chain (LC), non-glycosylated heavy chain (NGHC), heavy chain (HC), and thioether peak areas of ATL_5895 4-week thermal stability sample and 10× freeze-thaw study sample.

[0719] HTT exon-1 sandwich ELISA was performed to evaluate changes in ATL_5895 binding to 48Q HTT exon-1 after temperature stress {-80°C, 4°C, room temperature (21°C), and 40°C for 4 weeks} and 10× freeze-thaw cycles. Figure 27F It was shown that there was no significant change in binding to 48Q HTT exon-1 after temperature stress {-80°C, 4°C, room temperature (21°C), and 40°C for 4 weeks} and 10× freeze-thaw cycles compared to an isotype control antibody (ATL_5338-011).

[0720] Protein thermal shift and light scattering measurements were performed to determine the melting temperature (Tm1 / Tm2) and aggregation temperature (Tagg / Tonset) of ATL_5895 (see Materials and Methods above). Figure 27G The results of the thermal stability assay are shown and summarized in Table 20. These results indicate that ATL_5895 thermal stability parameters are within the typical range for IgG1 antibodies.

[0721]

[0722] Table 20: Melting temperature (Tm1 / Tm2) and aggregation temperature (Tagg) measured for 5 mg / mL of ATL_0005895 in 20 mM histidine-acetate, 150 mM NaCl pH 5.5.

[0723] To evaluate the solubility of ATL_5895 at different concentrations, a solubility assessment was performed (see the Materials and Methods section above). After ATL_5895 was concentrated to a maximum of 89.96 mg / mL using tangential flow filtration (TFF) and incubated at 21°C for 1 week, no increase in aggregation was observed ( Figure 27H , Figure 27I ). Monomer purity was consistently high (>97%) in all samples tested (Table 21, Table 22).

[0724]

[0725] Table 21: Summary of initial (T0) ATL_0005895 solubility study SEC-HPLC data showing monomer purity (%), high molecular weight (HMWS) species (%), and low molecular weight (LMWS) species (%).

[0726]

[0727] Table 22: Summary of SEC-HPLC data for ATL_0005895 solubility studies after 1 week at 21°C showing monomer purity (%), high molecular weight (HMWS) species (%), and low molecular weight (LMWS) species (%).

[0728] Example 17—Affinity-matured HTT antibody combination

[0729] Antibodies were generated using combinations of mutations present in the affinity matured variants ATL_6205, ATL_6202, ATL_6194 and / or ATL_6203 (see Example 11 above).

[0730] To evaluate the binding potency of affinity-matured antibodies to HTT exon 1 48Q protein, the antibodies were evaluated in a sandwich ELISA format for binding to human HTT exon 1 48Q captured by the anti-polyQ specific antibody, clone MW1 (Merck Millipore; #MABN2427; see "Materials and Methods" above). Lysozyme was used as a negative control antigen, and none of these antibodies showed discernible binding to this control. ATL5338 was used as a negative isotype control, and it did not show binding to HTT exon 1 48Q.

[0731] Table 23 shows the VHCDR sequences for each of the antibodies generated using the combinations of mutations present in the affinity matured variants, as well as the VH CDR sequences of ATL5895. The full VH and VL sequences for these antibodies are provided in Table 1.

[0732] Antibody VH CDR1 VH CR2 VH CDR3 ATL_6374 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYYGLDV ATL_6375 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYYGLDV ATL_6376 KAWMS RIKSGIDAGTTDYAAPVKG PPFYYYYGLDV ATL_6377 KAWMS RIKSGIDAGTTDYAAPVKG SPYYYYYGLDV ATL_6378 KAWMS RIKSGIDAGTTDYAAPVKG SPYYYYYGLDV ATL_5895 KAWMS RIKSGIDAGTTDYAAPVKG PPYYYYYGLDV

[0733] Table 23 - VH CDRs of antibodies generated using combinations of mutations present in affinity matured variants

[0734] Table 24 shows the EC50 values of sandwich ELISAs derived from newly generated antibodies bound to exon 1 48Q HTT. All of these antibodies showed binding to HTT, while control antibody ATL_5338 (isotype control, bound to fluorescein) did not bind to HTT. EC50 was calculated using a variable slope (four-parameter) nonlinear fit of the data. Each antibody produced had a similar low EC50 level. ATL_6375, ATL_6376, and ATL_6377 showed the maximum binding affinity of the test antibodies. The EC50 of ATL_5895 is shown in Table 10 (2.077017E-09M). The EC50 of comparative antibody ATL_0005059 / NI-302.8F1 is shown in Table 10 (1.725504E-08M).

[0735]

[0736] Table 24 - EC50 values derived from sandwich ELISA for the indicated antibodies.

[0737] Example 18—Mouse HTT ELISA

[0738] To assess the binding potency of the antibodies to murine HTT protein, the binding of ATL5895, ATL6376, and ATL6377 to murine HTT and human lysozyme (as a negative control antigen) was assessed in a direct ELISA format. ATL5338 was used as a negative isotype control.

[0739] Figure 28 Results of an indirect ELISA for antibody binding to mouse HTT are shown. ATL5895, ATL6376, and ATL6377 all showed binding to mouse HTT, while ATL5338 did not (isotype control). Lysozyme did not show discernible binding to any of the antibodies evaluated at concentrations less than 1 μM. These results indicate that ATL5895, ATL6376, and ATL6377 have cross-species reactivity to mouse HTT.

[0740] Example 19—Binding of affinity matured antibodies by biolayer interferometry

[0741] The binding interactions of ATL_5895 and affinity matured antibodies described in Examples 11 and 17 above were evaluated by bio-layer interferometry (BLI) using an Octet instrument. HTT exon 1 in the form of a biotinylated peptide (SEQ ID NO: 174) or mutant HTT exon 1 48Q (SEQ ID NO: 44) was loaded onto streptavidin or GST biosensors (Sartorius, 18-5019 and 18-5096), respectively. Subsequently, the sensor was immersed in a separate well containing the indicated concentration of mAb at 1000 rpm for 300 seconds to measure binding during the association phase. The binding response at the end of the association phase is reported for all mAbs.

[0742] Figure 29 shows the results of these experiments, demonstrating that the affinity matured antibodies bind to HTT exon 1 and mutant HTT exon 1 and display an increased binding propensity compared to the parental ATL_5895.

Claims

1. An isolated antibody or antibody fragment thereof, which specifically binds to huntingtin (HTT) or a fragment thereof, wherein the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, and a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3) or a sequence comprising one or two substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3), wherein the substitutions are at positions selected from positions 95 and 97, wherein the substitutions are selected from positions Y97F and P95S, wherein the position numbering is according to Kabat; iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and vi. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6) or an amino acid sequence comprising one, two, three or four amino acid substitutions compared to GSYAGTANV (SEQ ID NO: 6), wherein the substitutions are selected from A92G, A95E, and G89V, and Y91F, wherein the position numbering is according to Kabat.

2. The isolated antibody or fragment thereof according to claim 1, wherein: The isolated antibody or fragment thereof has improved binding to a mutated and / or aggregated HTT protein compared to a non-mutated and / or non-aggregated HTT protein, wherein the relative binding to mutated and / or aggregated HTT and non-mutated and / or non-aggregated HTT is measured by determining the ratio of the EC50 value of an HTT protein comprising a 25Q repeat in exon 1 or a fragment thereof to the EC50 value of an HTT protein comprising a 48Q repeat in exon 1 or a fragment thereof; wherein the isolated antibody or fragment thereof has a ratio of EC50 for binding to an HTT protein comprising a 25Q repeat in exon 1 or a fragment thereof and EC50 for binding to an HTT protein comprising a 48Q repeat in exon 1 or a fragment thereof of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or at least 2, as measured by sandwich ELISA.

3. The isolated antibody or antibody fragment of claim 1 or claim 2, wherein the antibody comprises: (a) a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3), and A light chain variable (VL) domain selected from the group consisting of: (1) A VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6); (2) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence VSYGGTENV (SEQ ID NO: 162); (3) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence VSFAGTANV (SEQ ID NO: 160); or (4) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 has the amino acid sequence VSYAGTANV (SEQ ID NO: 161); or (b) a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence PPFYYYYGLDV (SEQ ID NO: 158); and A light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 comprises the amino acid sequence VSYGGTENV (SEQ ID NO: 162); or (c) a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, wherein: i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1); ii. HCDR2 has the amino acid sequence RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2); iii. HCDR3 has the amino acid sequence SPYYYYYGLDV (SEQ ID NO: 157), and A light chain variable (VL) domain selected from the group consisting of: (1) A VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence VSYAGTANV (SEQ ID NO: 161); or (2) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, wherein: i. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); ii. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO: 5); iii. LCDR3 has the amino acid sequence VSYGGTENV (SEQ ID NO: 162).

4. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein the VH domain is a human VH domain, and / or wherein the antibody or fragment thereof has a VH domain framework sequence selected from the group consisting of: Framework sequence of ATL_0005895VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO:98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO:99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCIP(SEQ ID NO:172)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO:101), Framework sequence of ATL_0006376VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCVP(SEQ ID NO: 173)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO: 101), Framework sequence of ATL_0006199 VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCSP(SEQ ID NO: 169)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO: 101), and Framework sequence of ATL_0006200 VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVP(SEQ ID NO: 170)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO: 101), Framework sequence of ATL_0006202 VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCSP(SEQ ID NO: 171)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO: 101), or Framework sequence of ATL_006195 VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN(SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG(SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTP(SEQ ID NO: 180)-[CDRH3]-WGQGTTVTVSS(SEQ ID NO: 101).

5. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein the VL domain is a human VL domain and / or wherein the antibody or fragment thereof has the framework sequence of ATL_0005895 VL: QSALTQPRSVSGSPGQSVTISC (SEQ ID NO: 131)- [CDRL1]-WYQQHPGKAPKLMIY (SEQ ID NO: 133)-[CDRL2]-GVPDRFSGSKSGATASLTISGLQAEDEADYYC (SEQ ID NO: 138)-[CDRL3]-FGGTTKLTVL (SEQ ID NO: 139).

6. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein the HCDR1, HCDR2 and HCDR3 of the VH domain are in germline framework, and / or wherein the LCDR1, LCDR2, LCDR3 of the VL domain are in germline framework.

7. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein: The heavy chain variable domain comprises the amino acid sequence of any of the following: ATL5895VH (SEQ ID NO: 7), ATL6204 VH (SEQ ID NO: 7), ATL_6199VH (SEQ ID NO: 144), ATL6374 VH (SEQ ID NO: 148), ATL_6194VH (SEQ ID NO: 145), ATL_6375VH (SEQ ID NO: 145), ATL_6200VH (SEQ ID NO: 145), ATL_6202VH (SEQ ID NO: 146), ATL6203 VH (SEQ ID NO: 147), ATL_6205VH (SEQ ID NO: 148), ATL6376 VH (SEQ ID NO: 175), ATL6377 VH (SEQ ID NO: 176), ATL6195 VH (SEQ ID NO: 179), ATL6378 VH (SEQ ID NO: 176); and The light chain variable domain comprises the amino acid sequence of any of the following: ATL_5895VL (SEQ ID NO: 8), ATL_6199VL (SEQ ID NO: 8), ATL_6195VL (SEQ ID NO: 8), ATL_6002VL (SEQ ID NO: 8), ATL_6374VL (SEQ ID NO: 153), ATL_6375 (SEQ ID NO: 153), ATL6376 VL (SEQ ID NO: 153), ATL6378 VL (SEQ ID NO: 153), ATL_6194VL (SEQ ID NO: 154), ATL6377 VL (SEQ ID NO: 154), ATL6203 VL (SEQ ID NO: 154), ATL_6204VL (SEQ ID NO: 155), ATL_6205VL (SEQ ID NO: 156), ATL6202 VL (SEQ ID NO: 159).

8. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein: The heavy chain variable domain comprises the amino acid sequence of any one of ATL_5895VH (SEQ ID NO: 7), ATL_6376VH (SEQ ID NO: 175), ATL_6377 VH (SEQ ID NO: 176), or a sequence comprising at most 1, 2, or 3 mutations compared to any one of these sequences; and The light chain variable domain comprises the amino acid sequence of any one of ATL_5895VL (SEQ ID NO: 8), ATL_6376VL (SEQ ID NO: 153), ATL6377 VL (SEQ ID NO: 154), or a sequence comprising up to 1, 2 or 3 mutations compared to any one of these sequences.

9. The isolated antibody or fragment thereof according to any one of claims 1 to 8, wherein a. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 7)(ATL_5895VH); And the light chain variable domain comprises the amino acid sequence OR b. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 148)(ATL6374VH); And the light chain variable domain comprises the amino acid sequence OR c. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 145)(ATL6375VH); And the light chain variable domain comprises the amino acid sequence OR d. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPFYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 175)(ATL_6376VH); And the light chain variable domain comprises the amino acid sequence OR e. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPSPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 176)(ATL6377VH); And the light chain variable domain comprises the amino acid sequence OR f. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPSPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 176)(ATL_6378VH); And the light chain variable domain comprises the amino acid sequence OR g. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCSPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 144)(ATL_6199VH) And the light chain variable domain comprises the amino acid sequence OR h. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 145)(ATL6200 VH) And the light chain variable domain comprises the amino acid sequence OR i. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCSPPPFYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 146)(ATL_6202VH) And the light chain variable domain comprises the amino acid sequence OR j. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPSPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 147)(ATL6203 VH) And the light chain variable domain comprises the amino acid sequence OR k. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 7)(ATL_6204VH) And the light chain variable domain comprises the amino acid sequence OR 1. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 148)(ATL_6205VH) And the light chain variable domain comprises the amino acid sequence OR n. The heavy chain variable domain comprises an amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 145)(ATL6194 VH) And the light chain variable domain comprises the amino acid sequence OR o. The heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTPPPYYYYYGLDVWGQGTTVTVSS(SEQ ID NO: 179)(ATL_6195VH) And the light chain variable domain comprises the amino acid sequence OR p. The heavy chain variable domain comprises a variable domain comprising an amino acid sequence that has at least 95% sequence identity or comprises at most 1, 2 or 3 substitutions compared to any one of the heavy chain variable domains of (a) to (n), and / or the light chain variable domain comprises an amino acid sequence that has at least 90%, at least 95% sequence identity or comprises at most 1, 2, 3, 4 or 5 substitutions compared to any one of the light chain variable domains of (a) to (n).

10. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein: The HTT protein is human HTT or mouse HTT, and / or The isolated antibody or fragment thereof binds to a region located within exon 1 of HTT.

11. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein: The isolated antibody or fragment thereof binds to HTT or a fragment thereof comprising at least a portion of exon 1 with a lower EC50 value than a reference antibody as measured by sandwich ELISA, optionally wherein the HTT has a 25Q repeat or a 48 repeat in exon 1, and / or The isolated antibody or fragment thereof binds to HTT or a fragment thereof comprising at least a portion of exon 1 with an EC50 value of at most 15 nM, at most 12 nM, at most 10 nM or at most 5 nM, as measured using a sandwich ELISA, optionally wherein the HTT has a 25Q repeat or a 48 repeat in exon 1, and / or wherein the HTT or HTT fragment comprises the sequence of SEQ ID NO: 43, 44, 45 or 46, and / or wherein the sandwich ELISA is performed as described herein (Examples, Materials and Methods).

12. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein: The isolated antibody or fragment thereof has higher relative binding to mutated and / or aggregated HTT and non-mutated and / or non-aggregated HTT compared to a reference antibody (eg, such as ATL_0005059).

13. The isolated antibody or fragment thereof according to claim 11 or claim 12, wherein the reference antibody comprises: a. A heavy chain variable (VH) domain having the following CDRs: i. HCDR1 having the amino acid sequence NAWMN (SEQ ID NO: 35); ii. HCDR2 having the amino acid sequence HIRTQAEGGTSDYAAPVKG (SEQ ID NO: 36); iii. HCDR3 having the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3); b. a light chain variable (VL) domain having the following CDRs: i. LCDR1 having the amino acid sequence TGASSDVGTYDLVS (SEQ ID NO: 37); ii. LCDR2 having the amino acid sequence EVNKRPS (SEQ ID NO: 5); and iii. LCDR3 having the amino acid sequence CSYAGYSTV (SEQ ID NO: 38), Optionally, wherein the reference antibody is NI-302.8F1 described in US 11,401,325 B2.

14. An isolated antibody or fragment thereof according to any of the preceding claims, wherein the isolated antibody or fragment thereof binds to a mutant and / or aggregated HTT protein as determined by measuring immunoprecipitation of mutant HTT with the isolated antibody or fragment thereof, optionally wherein the mutant HTT is an HTT protein or fragment thereof comprising more than 35 glutamine residues in its polyQ tract, optionally comprising a fragment of exon 1.

15. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein: The isolated antibody or fragment thereof recognizes an epitope in a region corresponding to exon 1 of the HTT gene, and / or The isolated antibody or fragment thereof recognizes an epitope located in the polyP region of HTT, and / or wherein the isolated antibody or fragment thereof recognizes an epitope comprising the amino acid sequence QQQQPPPPPPPPP (SEQ ID NO: 47) or PQPQPPPPPPPPPPP (SEQ ID NO: 48), and / or The isolated antibody or fragment thereof is capable of crossing the blood-brain barrier, and / or The isolated antibody or fragment thereof is a bispecific antibody further comprising a region that binds to transferrin receptor.

16. An isolated antibody or fragment thereof according to any of the preceding claims, wherein the isolated antibody or fragment thereof increases phagocytosis of HTT protein or a fragment thereof comprising exon 1 containing 48Q repeats by cells, optionally wherein the cells are microglia, optionally iPSC-derived human microglia, optionally wherein the isolated antibody or fragment thereof increases phagocytosis of HTT protein or a fragment thereof by cells in a dose-dependent manner, and / or wherein the increased phagocytosis is measured by detecting phagocytosis of beads coated with HTT protein or fragments coated with a pH-sensitive fluorescent dye, and / or wherein the increased phagocytosis is measured as described herein (Materials and Methods).

17. An isolated antibody or fragment thereof according to any of the preceding claims, wherein the isolated antibody or fragment thereof reduces the aggregation rate of an HTT protein or fragment thereof comprising exon 1 containing a 48Q repeat in a cell-free assay, optionally wherein the reduced aggregation rate is measured using a FRASE assay, and / or wherein the reduced aggregation rate is measured as described herein (Materials and Methods), and / or wherein immunodepletion of a solution comprising the HTT protein or fragment thereof with the isolated antibody or fragment thereof results in a Δt50 for aggregation of the HTT protein or fragment thereof of at most 0.1, at most 0.2 or at most 0.3, and / or wherein the aggregation of the HTT protein or fragment thereof is measured in the presence of HTT fibrils and / or brain homogenate from one or more R6 / 2 mice.

18. An isolated antibody or fragment thereof according to any of the preceding claims, wherein the isolated antibody or fragment thereof reduces the aggregation of mutant HTT, wherein the aggregation of mutant HTT is measured as the presence and / or concentration of HTT aggregates in the brain of a Huntington's disease transgenic mouse model, optionally R6 / 1 mice, optionally wherein treatment of the mice with the isolated antibody or fragment thereof for 12 weeks or longer results in a statistically significant reduction in the concentration of HTT aggregates in the striatum and / or cortex.

19. The isolated antibody or fragment thereof according to any one of the preceding claims, wherein the antibody or fragment binds to mutant HTT in vivo.

20. The isolated antibody or fragment thereof according to claim 18 or claim 19, wherein the mutant HTT is an HTT protein or fragment thereof comprising more than 35 glutamine residues or 115 to 150 glutamine residues in its polyQ tract, optionally comprising a fragment of exon 1.

21. The isolated antibody or fragment thereof according to any one of claims 18 to 20, wherein the isolated antibody or fragment thereof does not reduce the level of non-mutated and / or non-aggregated HTT in the brain of a transgenic mouse model of Huntington's disease treated with the isolated antibody or fragment thereof, wherein the level of non-mutated and / or non-aggregated HTT is measured as the concentration of soluble and / or non-mutant HTT in the mouse, optionally wherein the mouse is an R6 / 1 mouse.

22. An isolated antibody or fragment thereof according to any of the preceding claims, wherein the antibody or fragment thereof retains a monomer percentage greater than 95% or greater than 97% after incubation at -80°C, 4°C, 21°C, 40°C for 4 weeks and / or after 10× freeze-thaw cycles; and / or wherein the antibody or fragment thereof binds to an HTT protein comprising HTT exon 1 and 48 glutamine residues in its polyQ tract after incubation at -80°C, 4°C, 21°C and 40°C for 4 weeks and / or 10× freeze-thaw cycles, as assessed by ELISA, optionally wherein the binding is not significantly different from the binding of the antibody to the HTT protein before incubation and / or 10× freeze-thaw cycles.

23. A DNA molecule or set of DNA molecules encoding an antibody or antibody fragment thereof according to any one of the preceding claims.

24. A vector or a set of vectors encoding a DNA molecule according to claim 23.

25. A host cell comprising the vector or a group of vectors according to claim 23.

26. A method for treating a disease or condition in a subject, comprising administering to the subject an effective amount of an isolated antibody or antibody fragment thereof according to any one of claims 1 to 22, optionally wherein the treatment prevents and / or reduces seeding and / or aggregation of mutant HTT in the subject.

27. The method of claim 26, wherein the treatment prevents and / or reduces aggregation of mutant HTT in the subject without reducing the level of non-mutated and / or non-aggregated HTT in the subject.

28. The method of claim 26 or claim 27, wherein the mutant HTT is an HTT protein comprising more than 35 glutamine residues or 115 to 150 glutamine residues in its polyQ tract, or a fragment thereof, optionally comprising a fragment of exon 1.

29. Use of the isolated antibody or antibody fragment thereof according to any one of claims 1 to 22 in the preparation of a medicament for treating a disorder or disease.

30. A composition comprising the isolated antibody or antibody fragment thereof according to any one of claims 1 to 22, optionally wherein the composition comprises a pharmaceutically acceptable excipient, vehicle or carrier, and / or wherein the composition is for use in treating a disease or disorder.

31. The method, use or composition for use according to any one of claims 26 to 30, wherein the disease or disorder is a neurodegenerative disease or disorder, optionally wherein the neurodegenerative disorder is Huntington's disease, Alzheimer's disease or frontotemporal dementia.

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