Anti-huntingtin antibody

Abstract

Antibodies that bind to the HTT protein and fragments thereof are described. Methods, including compositions and methods of treatment, comprising these antibodies are also described.

Description

[Technical Field]

[0001] This application claims priority from European Patent Application Publication No. 22212053.7, filed December 7, 2022, and British Patent Application Publication No. 2305916.5, filed April 21, 2023, the contents and elements of each of which are incorporated herein by reference for all purposes.

[0002] FIELD OF THE INVENTION 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 in the treatment of neurological disorders are also described. [Background technology]

[0003] background 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 clear genetic origin, the molecular and cellular mechanisms underlying HD are complex. Current treatments focus on clinical symptoms (e.g., monoamine depleting agents, thiazides / 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 symptoms caused by HD but are not disease-modifying. Oligonucleotide, gene, and cell therapies have progressed through clinical development, but there have been significant setbacks that may reflect delivery issues, the complexity of new modalities, a lack of differentiation between the pathological and physiological functions of HTT, and issues with patient acceptance of clinical trial design. There remains a need for improved treatments for Huntington's disease.

[0004] Alzheimer's disease (AD) is a neurodegenerative disorder that results in the progressive loss of brain cells. According to the 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 were living with dementia in 2015, and this number will reach 131.5 million by 2050. New therapies for AD are being actively sought to modify the disease course. Current candidates targeting brain beta-amyloid, tau, and innate immunity have occasionally demonstrated pharmacodynamic effects on pathological mechanisms in clinical trials, but to date, late-stage clinical trials have not demonstrated convincing disease modification. Improved disease-modifying therapies for AD remain needed (Golde TE (2022) Neurotherapeutics 19, 209-227).

[0005] The present invention has been devised in light of the above considerations. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present invention relates to novel, improved antibodies against HTT. By studying the immune responses of individuals who exhibit resilience to neurodegeneration despite increased risk of disease and comparing them with those with progressive disease, the inventors identified a cluster of related antibody heavy chains (VHs) that converged among resilient individuals. Target deconvolution showed that the convergent VHs could bind to HTT. The inventors further identified candidate antibodies derived from these identified VHs, resulting in antibodies predicted to slow or reverse neurodegeneration, identified using an unbiased approach to both antibody discovery and target identification. In particular, representative heavy chains were paired with appropriate light chains and expressed in an IgG1 format as antibodies designated herein as ATL5331, ATL5334, and ATL5335. These antibodies were developed to further improve their properties, including, but not limited to, improved binding potency, improved pharmacokinetic properties, reduced toxicity, and improved stability. These steps go beyond routine optimization of cohort diversity and the required analysis, extensive testing, simultaneous investigation of multiple beneficial mechanistic properties, and directed engineering to generate antibodies not found in naturally occurring populations. These novel antibodies with improved properties include those designated herein as ATL5895, ATL5901, and ATL5667, as well as 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 a huntingtin (HTT) protein 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), the substitutions being at positions selected from 95 and 97, and the substitutions being Y97 F and P95S, 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 containing 1, 2, 3 or 4 amino acid substitutions compared to GSYAGTANV (SEQ ID NO: 6), the substitutions being at positions selected from 92, 95, 89 and 91. The substitutions may be selected from A92G, A95E, G89V and Y91F, position numbering is according to Kabat.

[0008] Thus, the antibody may comprise the HCDR1, HCDR2 and HCDR3 of ATL5895, 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. 3 and LCDR1, LCDR2 and LCDR3 of ATL5895, 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.

[0009] Embodiments of any aspect may have any one or more of the following optional features.

[0010] The isolated antibody or fragment thereof may have improved binding to mutant and / or aggregated HTT protein compared to non-mutant and / or non-aggregated HTT protein, where the relative binding to mutant and / or aggregated and non-mutant and / or non-aggregated HTT is measured by determining the ratio of EC50 values ​​for an HTT protein or fragment thereof comprising a 25Q repeat in exon 1 and an HTT protein or fragment thereof comprising a 48Q repeat in exon 1. The isolated antibody or fragment thereof may have an EC50 ratio 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 for binding to an HTT protein or fragment thereof comprising a 25Q repeat in exon 1 and an HTT protein or fragment thereof comprising a 48Q repeat in exon 1, as measured by sandwich ELISA. HTT or HTT fragments containing a 48Q repeat may comprise the sequence of SEQ ID NO: 44 or 46, and / or HTT or HTT fragments containing a 25Q repeat may comprise the sequence of SEQ ID NO: 43 or 45. Sandwich ELISA may be performed as described herein (Examples, Materials and Methods).

[0011] In embodiments, the antibody fragment comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, where 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 or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, where 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 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).Exemplary antibodies according to these embodiments include ATL_5895, ATL_6194, ATL_6195, ATL_6374, ATL_6375, ATL_6199, ATL_6200, ATL_6204 and ATL6205.

[0012] In embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, where 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 PPFYYYYGLDV (SEQ ID NO: 158), and a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, where 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). Examples of antibodies according to these embodiments include ATL_6376 and ATL_6202.

[0013] In embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, where i. HCDR1h 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 SPYYYYYGLDV (SEQ ID NO: 157). In some such embodiments, the antibody or fragment comprises a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2, and LCDR3, where 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). Exemplary 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), and iii. LCDR3 has the amino acid sequence VSYGGTENV (SEQ ID NO:162). Exemplary antibodies according to these embodiments include ATL_6378.

[0014] The VH domain can be a human VH domain. The antibody or fragment thereof comprises the 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) The antibody or fragment thereof may have the framework sequence of ATL_0006200 VH (as well as ATL6374 VH and ATL_6194 VH): EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVP (SEQ ID NO: 170)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101) The antibody or fragment thereof may have the 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) The antibody or fragment thereof may have the framework sequence of ATL_0006203 VH (and ATL_5895 VH and ATL_6204 VH): EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCIP (SEQ ID NO: 172)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101) The antibody or fragment thereof may have the framework sequence of ATL_0006205 VH (and ATL_6375 VH, ATL_6376 VH, ATL6377 VH and ATL_6378 VH): EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCVP (SEQ ID NO: 173)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101) The antibody or fragment thereof may have the 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) may have:

[0015] In embodiments, the VL domain is a human VL domain. In embodiments, the antibody or fragment thereof comprises the VL domain framework sequence of ATL_0005895 VL: QSALTQPRSVSGSPGQSVTISC (SEQ ID NO: 131)-[CDRL1]-WYQQHPGKAPKLMIY (SEQ ID NO: 133)-[CDRL2]-GVPDRFSGSKSGATASLTISGLQAEDEADYYC (SEQ ID NO: 138)-[CDRL3]-FGTGTKLTVL (SEQ ID NO: 139) It has.

[0016] In embodiments, HCDR1, HCDR2 and HCDR3 of the VH domain are in a germline framework, hi embodiments, LCDR1, LCDR2 and LCDR3 of the VL domain are in a germline framework.

[0017] In embodiments, the heavy chain variable domain comprises the amino acid sequence of any of ATL_5895 VH (SEQ ID NO: 7), ATL_6204 VH (SEQ ID NO: 7), ATL_6199 VH (SEQ ID NO: 144), ATL_6374 VH (SEQ ID NO: 148), ATL_6194 VH (SEQ ID NO: 145), ATL_6375 VH (SEQ ID NO: 145), ATL_6200 VH (SEQ ID NO: 145), ATL_6202 VH (SEQ ID NO: 146), ATL_6203 VH (SEQ ID NO: 147), ATL_6205 VH (SEQ ID NO: 148), ATL_6376 VH (SEQ ID NO: 175), ATL_6377 VH (SEQ ID NO: 176), ATL_6195 VH (SEQ ID NO: 179), or ATL_6378 VH (SEQ ID NO: 176). In some such embodiments, the light chain variable domain comprises the amino acid sequence of any of ATL_5895 VL (SEQ ID NO: 8), ATL_6199 VL (SEQ ID NO: 8), ATL_6195 VL (SEQ ID NO: 8), ATL_6002 VL (SEQ ID NO: 8), ATL_6374 VL (SEQ ID NO: 153), ATL_6375 (SEQ ID NO: 153), ATL_6376 VL (SEQ ID NO: 153), ATL_6378 VL (SEQ ID NO: 153), ATL_6194 VL (SEQ ID NO: 154), ATL_6377 VL (SEQ ID NO: 154), ATL_6203 VL (SEQ ID NO: 154), ATL_6204 VL (SEQ ID NO: 155), ATL_6205 VL (SEQ ID NO: 156), or ATL_6202 VL (SEQ ID NO: 159).

[0018] In embodiments, the heavy chain variable domain comprises the amino acid sequence of any of ATL_5895 VH (SEQ ID NO: 7), ATL_6376 VH (SEQ ID NO: 175), ATL_6377 VH (SEQ ID NO: 176), or a sequence containing up to one, two, or three mutations compared to any of these sequences. In some such embodiments, the light chain variable domain comprises the amino acid sequence of any of ATL_5895 VL (SEQ ID NO: 8), ATL_6376 VL (SEQ ID NO: 153), ATL_6377 VL (SEQ ID NO: 154), or a sequence containing up to one, two, or three mutations compared to any of these sequences.

[0019] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_5895 VH).

[0020] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_5895 VL).

[0021] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 148) (ATL_6374 VH).

[0022] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6374 VL).

[0023] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6375 VH).

[0024] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6375 VL).

[0025] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPFYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 175) (ATL_6376 VH).

[0026] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6376 VL).

[0027] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 176) (ATL_6377 VH).

[0028] In some such embodiments, the light chain variable domain sequence comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6377 VL).

[0029] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 176) (ATL_6378 VH).

[0030] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6378 VL).

[0031] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCSPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 144) (ATL_6199 VH).

[0032] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6199 VL).

[0033] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6200 VH).

[0034] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6002 VL).

[0035] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCSPPPFYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 146) (ATL_6202 VH).

[0036] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 159) (ATL_6202 VL).

[0037] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 147) (ATL_6203 VH).

[0038] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6203 VL).

[0039] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_6204 VH).

[0040] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSFAGTANVFGTGTKVTVL (SEQ ID NO: 155) (ATL_6204 VL).

[0041] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 148) (ATL_6205 VH).

[0042] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 156) (ATL_6205 VL).

[0043] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6194 VH).

[0044] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6194 VL).

[0045] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 179) (ATL_6195 VH).

[0046] In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6195 VL).

[0047] In embodiments, the heavy chain variable domain comprises a variable domain comprising an amino acid sequence that has at least 95% sequence identity or contains up to 1, 2 or 3 substitutions compared to any one of the above heavy chain variable domains (SEQ ID NOs: 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 contains up to 1, 2, 3, 4 or 5 substitutions compared to any one of the above light chain variable domains (SEQ ID NOs: 8, 153, 154, 159, 155, 156).

[0048] In embodiments, the HTT protein is human or mouse HTT. In embodiments, the isolated antibody or fragment thereof binds to a region located within exon 1 of HTT. In embodiments, 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 compared to a reference antibody, as measured by sandwich ELISA. In embodiments, HTT has a 25Q repeat or a 48 repeat in exon 1. In embodiments, the isolated antibody or fragment thereof has improved binding to mutant and / or aggregated HTT protein compared to non-mutant and / or non-aggregated HTT protein. In embodiments, the relative binding to mutant and / or aggregated and non-mutant and / or non-aggregated HTT is measured by determining the ratio of the EC50 values ​​for an HTT protein or fragment thereof comprising a 25Q repeat in exon 1 and an HTT protein or fragment thereof comprising a 48Q repeat in exon 1. In embodiments, the HTT protein or fragment thereof is a fragment corresponding to exon 1. In embodiments, the EC50 is as measured by sandwich ELISA. In embodiments, the isolated antibody or fragment thereof has higher relative binding to mutated and / or aggregated and non-mutated and / or non-aggregated HTT compared to a reference antibody (e.g., ATL_0005059, etc.). In embodiments, the isolated antibody or fragment thereof has an EC50 ratio for binding to an HTT protein or fragment thereof comprising a 25Q repeat in exon 1 and an HTT protein or fragment thereof comprising a 48Q repeat in exon 1 of 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 ELISAs can be performed as described herein (Examples, Materials and Methods). Thus, the antibodies described herein can be capable of preferentially binding to mutant HTT, thereby reducing the seeding propensity of mutant HTT (i.e., reducing mutant HTT aggregation).

[0049] The isolated antibody or fragment thereof can increase phagocytosis by cells of HTT protein or fragments thereof comprising exon 1 containing a 48Q repeat. The cells can be microglial cells, optionally iPSC-derived human microglial cells. The isolated antibody or fragment thereof can increase phagocytosis by cells of HTT protein or fragments thereof comprising exon 1 containing a 48Q repeat in a dose-dependent manner. Increased phagocytosis can be measured by detecting phagocytosis of beads coated with HTT protein or fragments coated with a pH-sensitive fluorescent dye. Increased phagocytosis can be measured as described herein (Materials and Methods).

[0050] The isolated antibody or fragment thereof may reduce the aggregation rate of HTT protein or a fragment thereof containing exon 1 containing the 48Q repeat in a cell-free assay. The reduced aggregation rate may be measured using a FRASE assay. The reduced aggregation rate may be measured as described herein (Materials and Methods). Immunodepletion of a solution containing HTT protein or a fragment thereof using the isolated antibody or a fragment thereof may result in a delta t50 of at most 0.1, at most 0.2, or at most 0.3 for aggregation of HTT protein or a fragment thereof. Aggregation of HTT protein or a fragment thereof is measured in the presence of HTT fibrils (e.g., from recombinant HTT) and / or brain homogenate from one or more R6 / 2 mice.

[0051] In 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 embodiments, the reference antibody is NI-302.8F1, described in U.S. Patent No. 11,401,325.

[0052] In embodiments, the isolated antibody or fragment thereof binds to mutant and / or aggregated HTT protein as determined by measuring immunoprecipitation of mutant HTT with the isolated antibody or fragment thereof. In embodiments, the mutant HTT is an HTT protein or fragment thereof. In embodiments, the mutant HTT is an HTT fragment comprising exon 1. In embodiments, the mutant HTT is an HTT protein or fragment comprising more than 35 glutamine residues within its polyQ tract. Preferential binding to more aggregation-prone, and presumably more pathological, forms of HTT may advantageously inhibit the templating ability of aggregated HTT / mutant HTT and thus inhibit the progression of diseases associated with protein aggregation. For example, the isolated antibody or fragment described herein binds to an HTT protein comprising 110 CAG repeats (HTT 110As another example, mutant and / or aggregated HTT proteins may immunoprecipitate mutant and / or aggregated HTT proteins containing approximately 120 CAG repeats (e.g., extracted from cell or tissue samples, such as samples from R6 / 2 transgenic mice). R6 / 2 transgenic mice express the 5' end of the human HTT gene, including exon 1, which contains approximately 120 CAG repeats, and exhibit a neurological phenotype similar to that characteristic of HD in humans. As a further example, the isolated antibodies or fragments described herein may immunoprecipitate mutant and / or aggregated HTT proteins containing approximately 115-150 CAG repeats (e.g., extracted from cell or tissue samples, such as samples from R6 / 1 transgenic mice). R6 / 1 transgenic mice ubiquitously express a transgene containing the 5' end of a mutant human HTT gene, including approximately 1 kb of 5' UTR sequence, exon 1 (containing an expanded CAG repeat with 115-150 CAG repeats), and the first 262 bp of intron 1. R6 / 1 mice exhibit a progressive neurological phenotype that mimics many of the features of Huntington's disease (Mangiarini et al., Cell, 1996), including the accumulation of aggregates over time (Hansson et al., EJN, 2001).

[0053] In embodiments, the isolated antibody or fragment thereof reduces mutant HTT aggregation, where mutant HTT aggregation is measured as the presence and / or concentration of HTT aggregates in the brain of a transgenic mouse model of Huntington's disease. The mouse model may be an R6 / 1 mouse. Treatment of the mouse with the isolated antibody or fragment thereof for 12 weeks or more may result in a statistically significant reduction in the concentration of HTT aggregates in the striatum and / or cortex. In embodiments, the antibody or fragment binds to mutant HTT in vivo. The mutant HTT may be an HTT protein or fragment thereof comprising more than 35 glutamine residues or 115-150 glutamine residues in its polyQ tract, optionally comprising exon 1. In embodiments, the isolated antibody or fragment thereof does not reduce the level of non-mutant 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-mutant and / or non-aggregated HTT is measured as the concentration of soluble and / or non-mutant HTT in the mouse, and optionally, the mouse is an R6 / 1 mouse.

[0054] In embodiments, the antibody or fragment thereof maintains a monomer percentage of greater than 95% or greater than 97% after 4 weeks of incubation at -80°C, 4°C, 21°C, 40°C and / or after 10 freeze-thaw cycles. In embodiments, the antibody or fragment thereof binds to an HTT protein comprising exon 1 of HTT and the 48 glutamine residues in its polyQ tract as assessed by ELISA after 4 weeks of incubation at -80°C, 4°C, 21°C and 40°C and / or after 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.

[0055] In 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 sandwich ELISA. HTT may have a 25Q repeat or a 48 repeat in exon 1. HTT or an HTT fragment may comprise the sequence of SEQ ID NO: 43, 44, 45, or 46. Sandwich ELISA may be performed as described herein (Examples, Materials and Methods).

[0056] In embodiments, the isolated antibody or fragment thereof recognizes an epitope in a region corresponding to exon 1 of the HTT gene. In embodiments, the isolated antibody or fragment thereof recognizes an epitope located in the polyP region of HTT. In 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 embodiments, the isolated antibody or fragment thereof is capable of crossing the blood-brain barrier. In embodiments, the isolated antibody or fragment thereof is a bispecific antibody further comprising a region that binds to the transferrin receptor. The isolated antibody or antibody fragment according to any preceding embodiment is, for example, a bispecific antibody comprising a single-chain variable fragment (scFv) according to the first aspect and a binding moiety (e.g., scFv, nanobody, or aptamer) that binds to a brain receptor, such as the transferrin receptor.

[0057] In embodiments, the isolated antibody or antibody fragment comprises a single chain variable fragment (scFv) or a fragment antigen-binding region (Fab). In embodiments, the isolated antibody or antibody fragment comprises an antibody constant region. In embodiments, the isolated antibody comprises a whole antibody. Optionally, the whole antibody may be an IgG1 antibody.

[0058] In a further aspect, the present disclosure provides an isolated antibody VH domain of an isolated antibody or antibody fragment according to the previous aspect.

[0059] In a further aspect, the present disclosure provides an isolated antibody VL domain of the isolated antibody or antibody fragment according to the first or second aspect.

[0060] In a further aspect, the present disclosure provides a composition comprising an isolated antibody, antibody fragment, antibody VH domain or antibody VL domain according to the first or second aspect.

[0061] In a further aspect, the present disclosure provides a host cell transformed in vitro with a nucleic acid molecule encoding an antibody or antibody fragment thereof according to the first or second aspect.

[0062] In a further aspect, the present disclosure provides a method of producing an antibody or antibody fragment (e.g., comprising an antibody VH or VL domain) according to any embodiment of the first or second aspect, comprising culturing a host cell transformed in vitro with a nucleic acid molecule encoding the antibody or antibody fragment under conditions suitable for production of 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.

[0063] In a further aspect, the present disclosure provides a DNA molecule or set of DNA molecules encoding an antibody or antibody fragment thereof according to the first or second aspect.

[0064] In a further aspect, the present disclosure provides a vector or set of vectors encoding one or more DNA molecules according to the first or second aspect.

[0065] In a further aspect, the present disclosure provides a host cell comprising a vector or set of vectors according to the first or second aspect.

[0066] In a further aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated antibody or antibody fragment thereof according to the first or second aspect. The treatment may prevent and / or reduce seeding and / or aggregation of mutant HTT in the subject. The disease or disorder may be Huntington's disease, Alzheimer's disease, or frontotemporal dementia. In embodiments, the treatment comprises administering a further therapeutic agent simultaneously or sequentially with the isolated antibody or antibody fragment. In embodiments, the treatment prevents and / or reduces aggregation of mutant HTT in the subject (e.g., including the subject's brain) without reducing the level of non-mutant and / or non-aggregated HTT in the subject (e.g., including the subject's brain).

[0067] In a further aspect, the present disclosure provides the use of the isolated antibody or antibody fragment thereof according to the first or second aspect in the manufacture of a medicament for treating a disorder or disease, which may be Huntington's disease, Alzheimer's disease or frontotemporal dementia.

[0068] In a further aspect, the present disclosure provides a composition comprising the isolated antibody or antibody fragment thereof according to the first or second aspect. The composition may comprise a pharmaceutically acceptable excipient, vehicle, or carrier. The composition may be for use in treating a disease or disorder. The disease or disorder may be Huntington's disease, Alzheimer's disease, or frontotemporal dementia.

[0069] In a further aspect, the present disclosure relates to a method of diagnosing or monitoring the progression of a disease or disorder characterized by the presence of mutant and / or aggregated HTT in a patient, comprising exposing a sample obtained from the patient to an antibody or fragment thereof described herein.

[0070] In a further aspect, the present disclosure provides a method for determining the effect of a treatment (e.g., a drug, etc.) on the presence of aggregated HTT protein in a patient, the method comprising exposing a sample obtained from the patient to an antibody or fragment thereof described herein. The method of the foregoing aspect may comprise determining the level of HTT protein in the sample from the patient by detecting the antibody or fragment thereof, or binding between the HTT protein and the antibody or fragment thereof. The method may comprise comparing the determined level to a predetermined threshold or to a level determined for one or more control samples. The sample may be a blood sample or a cerebrospinal fluid sample.

[0071] In a further aspect, the present disclosure provides a method for preventing or reducing mutant HTT seeding and / or aggregation in a subject in need thereof, comprising administering to the subject a therapeutic amount of an isolated antibody or antibody fragment thereof.

[0072] The present disclosure also expressly includes combinations of the described aspects and preferred features, except where such combinations are expressly disallowed or explicitly avoided.

[0073] Diagram Overview BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention will now be described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0074] [Figure 1A] Schematic of the unbiased antibody discovery and target identification process. Antibody identification workflow. [Figure 1B] Schematic representation of the unbiased antibody discovery and target identification process. Alignment of seven antibodies discovered in an Alzheimer's patient cohort showing CDR3 sequence homology to a known HTT binder (NI-302.8F1 described in U.S. Patient No. 11,401,325 B2). [Figure 2A]Figure 1 shows a schematic representation of the phage display selection process. Generation of a functional scFv library. Appropriate phagemids containing a VL sub-library were designed and generated from a panel of healthy donors. VH from an AD-resilient individual was cloned upstream of the VL sub-library to generate a functional (fused VH and VL) scFv library. [Figure 2B] The phage display selection process is shown schematically. Phages representing a resilient library of AD scFvs were used for selection. Two to three rounds of selection were performed against immobilized HTT exon 1 antigen (human HTT exon 1 with a 48Q repeat (see Table 1 for the sequence)). In every selection round, the pool of scFvs was narrowed down to those with antigen-binding properties. Target antigen binding was confirmed using phage ELISA, and unique scFvs that bound were converted to IgG1 for antigen binding confirmation. [Figure 3A] 1 shows ELISA results for antibodies of the present disclosure. Phage ELISA for binding of phage-displayed antibody fragments corresponding to antibody sequences selected against human mutant HTT (mHTT) with a 48Q repeat (in each subsection of the plot, different colored bars represent different concentrations of mHTT): left to right: 10 μg / ml, 2 μg / ml, and 0.4 μg / ml). [Figure 3B] ELISA results for antibodies of the present disclosure: IgG1 antibody ELISA against human HTT exon 1 (48Q) protein showing strong dose-dependent binding for five of the seven IgG1 converted antibodies tested. [Figure 4A] The VH and phage-displayed VL pairings are shown. The VH sequence identified as converging in AD-resilient individuals is designated ATL_0005042 (ATL_5042). The homologous HTT-binding antibody is CA_0000274 (also designated ATL_0005059 or NI-302.8F1). [Figure 4B]Pairing of VH with phage-displayed VL is shown. After phage display selection and sequence analysis in HTT exon 1, the functionally paired VL (ATL_0005331-5335) was aligned to CA_0000274 VL. [Figure 5] Figure 1 shows the representation of peptides in peptide arrays used for HTT epitope mapping. PEPperMAP® epitope mapping and peptide screening of human IgG1 antibodies ATL_0005331, ATL_0005335, and ATL_0005566 was performed against the sequences of human, cynomolgus monkey, and mouse huntingtin exon 1. The huntingtin sequence was converted to linear 15-amino acid peptides with 14-amino acid peptide overlaps for high-resolution epitope data. [Figure 6] The results of epitope mapping and peptide screening of ATL_5331 are shown. Top panel: Intensity plot showing corrected intensity values ​​for human, cyno_1, cyno_2 (two distinct sequences likely representing cynomolgus monkey huntingtin protein sequences), and mouse huntingtin sequences sorted from the N-terminus of human huntingtin to the C-terminus of mouse huntingtin. IgG was tested at a fluorescence intensity value of 1 μg / ml plotted in green, while IgG was tested at a fluorescence intensity value of 10 μg / ml plotted in red, with 1000 added to each intensity value to facilitate visualization. The x-axis indicates HTT species. Antibody ATL_0005331 shows two distinct signals spanning HTT exon 1. Several antibody responses are observed against epitope-like sequence patterns formed by adjacent peptides with consensus motifs at each site. Bottom panel: Epitope mapping of ATL_0005331 based on human, cyno_1, cyno_2, and mouse huntingtin sequences. Amino acids from peptides derived from HTT exon 1 to which ATL_0005331 showed binding are shown in bold, and peptides showing the highest relative binding at each site in HTT exon 1 of different species are underlined. [Figure 7]The results of epitope mapping and peptide screening of ATL_5335 are shown. Top panel: Intensity plot showing corrected intensity values ​​for human, cyno_1, cyno_2, and mouse huntingtin sequences sorted from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein. IgG was tested at a fluorescence intensity value of 1 μg / ml plotted in green, while IgG was tested at a fluorescence intensity value of 10 μg / ml plotted in red, with 2000 added to each intensity value to facilitate visualization. The x-axis indicates HTT species. Antibody ATL_0005335 shows two distinct signals spanning HTT exon 1. Several antibody responses are observed against epitope-like sequence patterns formed by adjacent peptides with consensus motifs at each site. Bottom panel: Epitope mapping of ATL_0005335 based on human, cyno_1, cyno_2, and mouse huntingtin sequences. Amino acids from peptides derived from HTT exon 1 to which ATL_0005335 showed binding are shown in bold, and peptides showing the highest relative binding at each site in HTT exon 1 of different species are underlined. [Figure 8]The results of epitope and peptide screening of ATL_5566 are shown. Top panel: Intensity plot showing corrected intensity values ​​for human, cyno_1, cyno_2, and mouse huntingtin sequences sorted from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein. IgG was tested at a fluorescence intensity value of 1 μg / ml plotted in green, while IgG was tested at a fluorescence intensity value of 10 μg / ml plotted in red, with 1000 added to each intensity value to facilitate visualization. The x-axis indicates HTT species. Antibody ATL_0005566 shows two distinct signals spanning HTT exon 1. Several antibody responses are observed against epitope-like sequence patterns formed by adjacent peptides with consensus motifs at each site. Bottom panel: Epitope mapping of ATL_0005566 based on human, cyno_1, cyno_2, and mouse huntingtin sequences. Amino acids from peptides derived from HTT exon 1 to which ATL_0005566 showed binding are shown in bold, and peptides showing the highest relative binding at each site in HTT exon 1 of different species are underlined. [Figure 9] The workflow for the 3-week stability study, low pH hold, and freeze-thaw cycle test is shown. The six indicated antibodies were normalized to 5 mg / ml and then used for the 3-week stability study, freeze-thaw study, or low pH hold test. Quality control analyses, 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, were performed. [Figure 10A] Figure 1 shows the results of a 3-week stability study or freeze-thaw cycling: SEC-HPLC. SEC-HPLC chromatograms of the three indicated antibodies (ATL_5331, ATL_5334, ATL_5335) after 3 weeks of incubation at the indicated temperatures (-80°C, +4°C, +21°C, +40°C) or five freeze-thaw cycles. [Figure 10B]Figure 1 shows the results of a 3-week stability study or freeze-thaw cycling: SEC-HPLC. SEC-HPLC chromatograms of the three indicated antibodies (ATL_5555, ATL_5556, ATL5557) after 3 weeks of incubation at the indicated temperatures (-80°C, +4°C, +21°C, +40°C) or five freeze-thaw cycles. [Figure 11] Results of a low pH retention study are shown. SEC-HPLC. Antibodies were prepared at a concentration of 5 mg / ml (PBS). Acetic acid was added dropwise to pH 3.5, and Tris base (pH 11) was added to neutralize the solution to pH 7.2-7.4. Chromatograms of ATL_5331 and ATL_5335 at 0, 15, 30, 60, or 120 minutes after pH neutralization. [Figure 12A] Figure 1 shows the results of charge heterogeneity assessment by cIEF. pI (isoelectric point) of each of the six antibodies before the 3-week stability study. [Figure 12B-1] Figure 1 shows the results of charge heterogeneity assessment by cIEF: the respective pIs of the two antibodies after a 3-week stability study at the indicated temperatures. [Figure 12B-2] Figure 1 shows the results of charge heterogeneity assessment by cIEF: the respective pIs of the two antibodies after a 3-week stability study at the indicated temperatures. [Figure 12B-3] Figure 1 shows the results of charge heterogeneity assessment by cIEF. The respective pIs of two antibodies after a 3-week stability study at the indicated temperatures. C-1 to C-3. The respective pIs of six antibodies after a 3-week stability study at -80°C or five freeze-thaw cycles (5xFT). [Figure 12C-1] Figure 1 shows the results of charge heterogeneity assessment by cIEF. Respective pI of two antibodies after a 3-week stability study at -80°C or five freeze-thaw cycles (5xFT). [Figure 12C-2] Figure 1 shows the results of charge heterogeneity assessment by cIEF. Respective pI of two antibodies after a 3-week stability study at -80°C or five freeze-thaw cycles (5xFT). [Figure 12C-3] Figure 1 shows the results of charge heterogeneity assessment by cIEF. Respective pI of two antibodies after a 3-week stability study at -80°C or five freeze-thaw cycles (5xFT). [Figure 13] Figure 1 shows the results of an HTT pharmacokinetic (PK) study: ATL_0005335 serum levels in mice detected by ELISA at 0, 1, 4, 8, 24, 72 and 144 hours after treatment with 10 or 20 mg / Kg. [Figure 14] 1 illustrates a schematic of the workflow for triaging a panel of antibody variants to identify a lead antibody. [Figure 15] ELISA of recombinant HTT exon 1 (48Q GST - i.e., HTT exon-1 48Q with a glutathione S-transferase tag used to purify the antigen by affinity chromatography), bars, and thermal stability analysis of mutant antibodies (dots) are shown. Melting temperatures were measured using SYPRO Orange. [Figure 16A] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 1. [Figure 16B] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 1. [Figure 16C] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 1. [Figure 16D] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 2. [Figure 16E]HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 2. [Figure 16F] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 2. [Figure 16G] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 2. [Figure 16H] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 3. [Figure 16I] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 3. [Figure 16J] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 25Q ELISA repeat 3. [Figure 16K] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 1. [Figure 16L]HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 1. [Figure 16M] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 1. [Figure 16N] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 1. [Figure 16O] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 2. [Figure 16P] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 2. [Figure 16Q] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 2. [Figure 16R] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 2. [Figure 16S]HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 3. [Figure 16T] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 3. [Figure 16U] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 3. [Figure 16V] HTT sandwich ELISA results for ATL_5895, ATL_5901, ATL5567, and ATL_5059 are shown (top row of table, as indicated within each panel). HTT exon 1 48Q ELISA repeat 3. [Figure 17A] The results of HTT immunoprecipitation are shown. Immunoprecipitation of HTT by ATL_5335 was tested in the U-2 OS cell line, which expresses 110 CAG repeats, resulting in a high molecular weight HTT species (designated "HTT110") and the parental (control) line (designated "HTTWT"). Anti-HTT 1C2 antibody (Sigma Aldrich MAB1574) was used for detection. [Figure 17B] Results of HTT immunoprecipitation are shown. Immunoprecipitation of HTT by ATL_0005895, ATL_0005901, and ATL_0005567 was tested using homogenates derived from R6 / 2 mouse brain tissue (denoted "R62 brain homog") or non-transgenic brain tissue (denoted "non-Tg brain homog"). Anti-HTT MW8 antibodies (MABN2529 Sigma) and 1C2 (MAB1574 Sigma-Aldrich) were used for detection. [Figure 17C]Figure 1 shows the results of HTT immunoprecipitation. Immunoprecipitation of HTT by ATL_0005895 was tested using homogenates derived from human Huntington's disease brain tissue. Anti-HTT antibodies HD1 and MW1 (MABN2427 Millipore) were used for detection. [Figure 17D] Results of HTT immunoprecipitation are shown. Densitometric analysis of the blot in Figure 17C performed using Image J analysis software (NIH). [Figure 18A] Figure 1 shows the results of a FRET-based mHTT (FRASE) assay to assess the ability of antibodies to bind seed-competent HTT (48Q) species and affect HTT aggregation. The aggregation rates (Δt values) of seed-competent HTT (mHTT) in the presence of fibrils produced from recombinant HTT after immunodepletion of the species using the indicated antibodies (ATL5895, ATL5901, MW8, and MW1) are shown. Data shown are representative examples from three biological replicates. Error bars represent + / - SD from n=3 technical replicates in a single biological replicate. [Figure 18B]Figure 1 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. The aggregation rates (Δt values) of the amount of R6 / 2 brain added as seed-competent HTT, designated "seed" in the figure, are shown in the presence of brain homogenates from R6 / 2 mouse brains after seed immunodepletion using the indicated antibodies. Seed immunodepletion was performed using the indicated antibodies (ATL5895, ATL5901, MW8, and MW1) on protein G beads (R6 / 2 "seeds" were incubated with antibody on protein G beads at 4°C for 1 hour, followed by removal of the antibody-bound beads, removal of the seed crystals if antibody was bound, and the remaining solution was used in the FRASe aggregation assay). Data shown are representative of three biological replicates, each using brains from a different R6 / 2 mouse. Error bars represent + / - SD from n=3 technical replicates within a single biological replicate. These data show that immunodepletion of seeds with antibodies ATL_0005895 and ATL_0005901 reduced the ability of HTT seeds from both sources (fibrils from recombinant HTT and brain homogenates from R6 / 2 mice) to increase the in vitro aggregation rate. 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. [Figure 19] Figure 1 shows a dose-dependent increase in phagocytosis of 48QHTT-coated beads by iPSC-derived microglia in the presence of ATL5895. Uptake of Q48 HTT exon 1-coated pHrodo™ red beads by induced pluripotent stem cell (iPSC) microglia was measured in the presence of ATL5895 or ATL5338, a human IgG1 isotype control antibody that binds fluorescein. ATL5895 increases the total red area signal over time compared to the isotype control antibody. The graph shows the area under the curve (AUC) calculated from the total red fluorescent signal per well over 4 hours of reaction versus antibody concentration (log nM). [Figure 20A]

[0023] Figure 1 shows the results of an in vivo pharmacokinetic (PK) study of antibodies of the present disclosure. Serum levels of ATL_0005567 and 5901 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. [Figure 20B]

[0023] Figure 1 shows the results of an in vivo pharmacokinetic (PK) study of antibodies of the present disclosure. Serum levels of ATL_0005895 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 are shown. [Figure 20C]

[0023] Figure 1 shows the results of an in vivo pharmacokinetic (PK) study of an antibody of the present disclosure. Shown are ATL_0005895 cerebrospinal fluid (CSF) levels in mice detected by ELISA 4 hours and 144 hours after treatment with 1, 10, and 60 mg / kg antibody by IP injection. ATL_0005895 1 mg / kg and 144 hour 10 mg / Kg CSF levels were below the limit of quantitation of the assay. [Figure 21A] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6199. Absorbance was measured at 450 nm. [Figure 21B] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6200. Absorbance was measured at 450 nm. [Figure 21C] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6202. Absorbance was measured at 450 nm. [Figure 21D] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6203. Absorbance was measured at 450 nm. [Figure 21E] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6204. Absorbance was measured at 450 nm. [Figure 21F] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6205. Absorbance was measured at 450 nm. [Figure 21G] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6194. Absorbance was measured at 450 nm. [Figure 21H] Shown are the results of an indirect ELISA for binding of the indicated antibodies to HTT exon 1 48Q or lysozyme control. ATL_6195. Absorbance was measured at 450 nm. [Figure 22A] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6199. Absorbance was measured at 450 nm. [Figure 22B] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6200. Absorbance was measured at 450 nm. [Figure 22C] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6202. Absorbance was measured at 450 nm. [Figure 22D] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6203. Absorbance was measured at 450 nm. [Figure 22E] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6204. Absorbance was measured at 450 nm. [Figure 22F]Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6205. Absorbance was measured at 450 nm. [Figure 22G] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 2 was performed using an antibody concentration of 400 nM and the results are shown. ATL_6199. Absorbance was measured at 450 nm. [Figure 22H] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 2 was performed using an antibody concentration of 400 nM, and the results are shown. ATL_6200. Absorbance was measured at 450 nm. [Figure 22I] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 2 was performed using an antibody concentration of 400 nM, and the results are shown. ATL_6202. Absorbance was measured at 450 nm. [Figure 22J] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 2 was performed using an antibody concentration of 400 nM and the results are shown. ATL_6203. Absorbance was measured at 450 nm. [Figure 22K] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 2 was performed using an antibody concentration of 400 nM and the results are shown. ATL_6204. Absorbance was measured at 450 nm. [Figure 22L] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 2 was performed using an antibody concentration of 400 nM and the results are shown. ATL_6205. Absorbance was measured at 450 nm. [Figure 22M]Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6194. Absorbance was measured at 450 nm. [Figure 22N] Shown are the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Repeat 1 was performed using an antibody starting concentration of 133 nM and the results are shown. ATL_6195. Absorbance was measured at 450 nm. [Figure 22O] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. ATL_6194. Absorbance was measured at 450 nm. [Figure 22P] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. ATL_6195. Absorbance was measured at 450 nm. [Figure 23A] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. ATL_6183. Absorbance was measured at 450 nm. [Figure 23B] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. ATL_6184. Absorbance was measured at 450 nm. [Figure 23C] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. ATL_6185. Absorbance was measured at 450 nm. [Figure 23D] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. ATL_6186. Absorbance was measured at 450 nm. [Figure 23E] Figure 1 shows the results of a sandwich ELISA for binding of the indicated antibodies to HTT exon 1 Q48. Control antibody ATL_5338. Absorbance was measured at 450 nm. [Figure 24A-1]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity-determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of HFW1, HCDR1, HFW2, HCDR2, HFW3, HCDR3, and HFW4 for the indicated antibodies as defined by Kabat. The heavy chain sequences of the antibodies described in Examples 1-10, 13-16, and 18 are shown. [Figure 24A-2]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity-determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of HFW1, HCDR1, HFW2, HCDR2, HFW3, HCDR3, and HFW4 for the indicated antibodies as defined by Kabat. The heavy chain sequences of the antibodies described in Examples 1-10, 13-16, and 18 are shown. [Figure 24A-3]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity-determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of HFW1, HCDR1, HFW2, HCDR2, HFW3, HCDR3, and HFW4 for the indicated antibodies as defined by Kabat. The heavy chain sequences of the antibodies described in Examples 11-12 are shown. [Figure 24A-4]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity-determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of HFW1, HCDR1, HFW2, HCDR2, HFW3, HCDR3, and HFW4 for the indicated antibodies as defined by Kabat. The heavy chain sequences of the antibodies described in Examples 11-12 are shown. [Figure 24A-5]

[0023] Figures 1A-1C show the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat.

[0024] Figures 1A-1C show the sequences of HFW1, HCDR1, HFW2, HCDR2, HFW3, HCDR3, and HFW4 for the indicated antibodies as defined by Kabat.

[0025] Figures 1B-1C show the heavy chain sequences of the antibodies described in Examples 11-12.

[0026] Figures 1C-1D show the heavy chain sequences of the antibodies described in Examples 17-18. [Figure 24B-1]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of LFW1, LCDR1, LFW2, LCDR2, LFW3, LCDR3, and LFW4 for the indicated antibodies as defined by Kabat. The light chain sequences of the antibodies described in Examples 1-10, 13-16, and 18 are shown. [Figure 24B-2]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of LFW1, LCDR1, LFW2, LCDR2, LFW3, LCDR3, and LFW4 for the indicated antibodies as defined by Kabat. The light chain sequences of the antibodies described in Examples 1-10, 13-16, and 18 are shown. [Figure 24B-3]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of LFW1, LCDR1, LFW2, LCDR2, LFW3, LCDR3, and LFW4 for the indicated antibodies as defined by Kabat. The light chain sequences of the antibodies described in Examples 11-12 are shown. [Figure 24B-4]

[0023] Figure 1 shows the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat. The sequences of LFW1, LCDR1, LFW2, LCDR2, LFW3, LCDR3, and LFW4 for the indicated antibodies as defined by Kabat. The light chain sequences of the antibodies described in Examples 11-12 are shown. [Figure 24B-5]

[0023] Figures 1A-1C show the sequences of the framework (FW) regions and complementarity determining regions (CDRs) of the antibodies of the present disclosure as defined by Kabat.

[0024] Figures 1A-1C show the sequences of the LFW1, LCDR1, LFW2, LCDR2, LFW3, LCDR3, and LFW4 for the indicated antibodies as defined by Kabat.

[0025] Figures 1B-1C show the light chain sequences of the antibodies described in Examples 11-12.

[0026] Figures 1C-1D show the light chain sequences of the antibodies described in Examples 17-18. [Figure 25A]Results of live animal PET / CT scans using radiolabeled antibodies and gamma counting assays are shown. Percentage of injected dose per gram of blood (%ID) detected by gamma counting from 0 to 168 hours post-dose in 11-12 week old cohorts of C57BL / 6J and R6 / 1 mice. [Figure 25B] Results of live animal PET / CT scans using radiolabeled antibodies and gamma counting assays are shown. Percentage of injected dose per gram of blood (%ID) detected by gamma counting from 0 to 168 hours post-dose in 14-15 week old cohorts of C57BL / 6J and R6 / 1 mice. [Figure 25C] Results of live animal PET / CT scans using radiolabeled antibodies and gamma counting assays are shown. Ex vivo biodistribution of 89Zr-Df-ATL5895 in 11-12 week old C57BL / 6J mice (left) and R6 / 1 mice (right) 168 hours after administration, as assessed by gamma counting analysis. [Figure 25D] Results of live animal PET / CT scans using radiolabeled antibodies and gamma counting assays are shown. Ex vivo biodistribution of 89Zr-Df-ATL5895 in 14-15 week old C57BL / 6J mice (left) and R6 / 1 mice (right) 168 hours after administration, as assessed by gamma counting analysis. [Figure 25E] Results of live animal PET / CT scans and gamma counting assays using radiolabeled antibodies are shown. Brain biodistribution determined by PET / CT imaging of 89Zr-Df-ATL5895 over 168 hours in 14-15 week old cohorts of C57BL / 6J and R6 / 1 mice. Representative coronal, sagittal, and transverse PET / CT images of WT and R6-1 mice. [Figure 25F]Results of live animal PET / CT scans and gamma counting assays using radiolabeled antibodies are shown. Brain biodistribution determined by PET / CT imaging of 89Zr-Df-ATL5895 over 168 hours in 14-15 week old cohorts of C57BL / 6J and R6 / 1 mice. [Figure 26A]

[0023] Figure 1 shows the results of an immunoassay (Mesoscale Discovery (MSD) assay) to assess the effect of ATL_5895 (ATLX_1095) on HTT aggregate burden in the striatum and cortex of R6 / 1 mice. Aggregated HTT increases over time in the striatum and cortex. [Figure 26B]

[0039] Figure 1 shows the results of an immunoassay (Mesoscale Discovery (MSD) assay) to assess the effect of ATL_5895 (ATLX_1095) on HTT aggregate burden in the striatum and cortex of R6 / 1 mice. Treatment of R6 / 1 mice with ATL_5895 (ATLX-1095) for 12 weeks resulted in a statistically significant reduction in HTT aggregates (MW8 / 4C9+) in the striatum and cortex. [Figure 26C] Figure 1 shows the results of an immunoassay (Mesoscale Discovery (MSD) assay) to assess the effect of ATL_5895 (ATLX_1095) on HTT aggregate burden in the striatum and cortex of R6 / 1 mice. ATL_5895 (ATLX-1095) treatment does not affect levels of mutant soluble HTT over time. [Figure 26D] Figure 1 shows the results of an immunoassay (Mesoscale Discovery (MSD) assay) to assess the effect of ATL_5895 (ATLX_1095) on HTT aggregate burden in the striatum and cortex of R6 / 1 mice. ATL_5895 (ATLX-1095) does not affect the levels of endogenous mouse HTT over time. [Figure 27A] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). SEC-HPLC chromatogram of the ATL_5895-002 4-week thermal stability study sample. The sample was run on a Zorbax GF-250 SEC-HPLC column (Agilent). [Figure 27B]The results of a manufacturability study of ATL_5895 (ATLX_1095) are shown. SEC-HPLC chromatogram of ATL_5895-002 10x freeze-thaw sample. The sample was run on a TSKgel G3000SWxl column (TOSOH Bioscience). [Figure 27C] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). cIEF electropherogram of the ATL_5895-002 4-week thermal stability study sample. [Figure 27D] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). ATL_5895-002 cIEF electropherogram of a 10-cycle freeze-thaw sample versus a non-stressed control sample. [Figure 27E] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). ATL_5895-002 thermal stability study sample and reduced CE-SDS trace for 10 freeze-thaw cycles. [Figure 27F] Results of a manufacturability study of ATL_5895 (ATLX_1095) are shown. ATL_5895-002 HTT exon-1 sandwich ELISA of 4-week thermal stability study samples and 10 freeze-thaw cycle samples. [Figure 27G] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). Melting temperatures (Tm1 / Tm2) and aggregation temperatures (Tagg) measured for ATL_0005895-002 at 5 mg / mL in 20 mM histidine-acetate, 150 mM NaCl pH 5.5. [Figure 27H] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). 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. [Figure 27I] Figure 1 shows the results of a manufacturability study of ATL_5895 (ATLX_1095). SEC-HPLC chromatograms of ATL_5895 solubility study samples after 1 week of incubation at 21°C at 11.90 mg / mL, 23.91 mg / mL, 44.95 mg / mL, and 89.96 mg / mL. [Figure 28-1] Figure 1 shows the results of a binding study of ATL5895, ATL6376, and ATL6377 to mouse HTT protein using direct ELISA. Human lysozyme was used as a control antigen. ATL5338 was used as a negative isotype control. [Figure 28-2] Figure 1 shows the results of a binding study of ATL5895, ATL6376, and ATL6377 to mouse HTT protein using direct ELISA. Human lysozyme was used as a control antigen. ATL5338 was used as a negative isotype control. [Figure 29A]

[0023] Figure 1 shows the results of a binding study of affinity-optimized antibodies to HTT exon 1 or mutant HTT exon 1 48Q. HTT exon 1 epitope peptide binding responses measured by Octet for the indicated mAbs tested at 15 nM each. The binding responses confirm that the mAbs bind to HTT exon 1 and show increased binding propensity compared to the parent ATL_5895. [Figure 29B]

[0023] Figure 1 shows the results of a binding study of affinity-optimized antibodies to HTT exon 1 or mutant HTT exon 1 48Q. HTT exon 1 epitope peptide binding responses measured by Octet for the indicated mAbs tested at 15 nM each. The binding responses confirm that the mAbs bind to HTT exon 1 and show increased binding propensity compared to the parent ATL_5895. [Figure 29C]

[0023] Figure 1 shows the results of a binding study of affinity-optimized antibodies to HTT exon 1 or mutant HTT exon 1 48Q. Mutant HTT exon 1 binding responses measured by Octet for the indicated mAbs tested at 25 nM each. The binding responses confirm that the mAbs bind to HTT exon 1 and show increased binding propensity compared to the parent ATL_5895. DETAILED DESCRIPTION OF THE INVENTION

[0075] Detailed Description of the Invention Aspects and embodiments of the present invention will now be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0076] Disclosed herein are antibodies and fragments thereof capable of specifically binding to huntingtin (HTT) protein or fragments thereof. The present disclosure refers to the antibodies described herein using references designated as "ATL_000xxxx," "ATL_xxxx," or "xxxx," where "xxxx" is a four-digit reference number specific to the antibody described herein. The above designations are all used interchangeably to refer to the same antibody or portion thereof (e.g., the VH, VL, or portion thereof of the antibody). For example, antibody ATL_0005895 is referred to interchangeably herein as ATL_5895 and 5895.

[0077] As used herein, an antibody capable of "specifically binding" to a target or capable of "specifically binding" to a target is an antibody that can bind through the association of an epitope recognition site with an epitope within the target. This is different from non-specific binding, such as Fc-mediated binding, ionic and / or hydrophobic interactions. In other words, antibodies that specifically bind to a target generally recognize and bind to specific protein structures therein, rather than proteins themselves, and are widely distributed throughout the CNS.

[0078] HTT is a soluble, 3144 amino acid (384 kda) protein (non-expanded form) widely distributed throughout the central nervous system (CNS) and associated with the neurodegenerative disorder Huntington's disease (HD). HD is caused by an expansion of the trinucleotide repeat CAG in exon 1 of HTT, which encodes a polyglutamine (polyQ) tract near the N-terminus of toxic mutant huntingtin (mHTT). In healthy individuals, the length of the CAG repeat typically ranges from 9 to 35 CAG repeats, but repeat numbers greater than 40 result in disease manifestation. A CAG repeat length of 36 to 39 is associated with reduced penetrance, resulting in some individuals developing HD and others remaining disease-free.

[0079] The expanded polyQ tract in mutant huntingtin leads to protein misfolding, resulting in 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. Additionally, mutant aggregates exhibit "seeding" potential; i.e., these proteins spontaneously aggregate when assessed, for example, in cell-free assays. The aggregates accelerate the aggregation rate of mHTT and cause the spread of pathological HTT in the CNS. Advantageously, the antibodies of the present disclosure can bind to toxic extracellular mHTT and prevent its propagation through removal of aggregated mHTT and / or inhibition of mHTT's seeding ability. The human gene encoding HTT (Gene ID: 3064) is located at 4p16.3 and is large, spanning 180 kb and consisting of 67 exons. Reference non-human HTT amino acid and coding sequences are available in public databases.

[0080] The reference amino acid sequence of human HTT exon 1 is provided below as "wild-type huntingtin (HTT) exon 1" or "mutant huntingtin (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 sequence 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 (HTT) exon 1" or "mutant huntingtin (HTT) exon 1" below. In some embodiments, the antibodies described herein can specifically bind to a peptide or protein, or a fragment thereof, having or comprising the amino acid sequence of "wild-type huntingtin (HTT) exon 1" or "mutant huntingtin (HTT) exon 1" (including a full-length HTT protein comprising said sequence or a fragment of said protein comprising said sequence).

[0081] In some embodiments, the antibodies described herein can specifically bind to an HTT protein or protein fragment comprising or consisting of the amino acid sequence of an HTT variant. In some embodiments, the HTT variant protein or fragment comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or at least 99% identity to "wild-type huntingtin (HTT) exon 1" or "mutant huntingtin (HTT) exon 1." In some embodiments, the antibodies disclosed herein can specifically bind to an HTT fragment comprising at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the HTT amino acid sequence or HTT variant sequence.

[0082] In some embodiments, the antibodies and fragments of the present disclosure bind to a region in exon 1 of HTT. Exon 1 of HTT can contain an expanded polyQ repeat caused by an expanded CAG repeat (e.g., more than 35-40 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 manifestation can occur. Thus, the term "mutant HTT" (mHTT), as used herein, 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. An HTT protein or fragment 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 an expanded polyQ tract (also called "high molecular weight HTT"), e.g., HTT with 36 or more glutamines.

[0083] 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 may have a polyQ tract containing 25 glutamines. Human HTT exon 1 with a polyQ tract containing 25 glutamines may have the following sequence (referred to as "wild-type huntingtin (HTT) exon 1"): >Human_HTT_Exon_1_25Q (SEQ ID NO: 43) [ka]

[0084] In some embodiments, HTT exon 1 has a polyQ tract containing more than 40 glutamines. In some embodiments, the polyQ tract includes 48 glutamines. For example, human HTT exon 1 may have a polyQ tract containing 48 glutamines. Human HTT exon 1 with a polyQ tract containing 48 glutamines may have the following sequence (referred to as "mutant huntingtin (HTT) exon 1"): >Human_HTT_Exon_1_48Q (SEQ ID NO: 44) [ka]

[0085] The binding regions 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 antibodies bind to an epitope within the polyP and / or polyQ / polyP region of HTT. In some embodiments, the epitope comprises or is contained within the amino acid sequence QQQQPPPPPPPPPPP (SEQ ID NO: 47) or amino acid sequence PQPQPPPPPPPPPPP (SEQ ID NO: 48) of human HTT or the corresponding region in a homologous protein. Binding regions of exemplary antibodies of the present disclosure for various HTT proteins are shown in Figures 6-8 (bolded regions). In embodiments, the binding regions of the antibodies described herein are located in any of the bolded regions of Figure 6, Figure 7, or Figure 8. The region in exon 1 of HTT has been 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 embodiments, the antibody binds to an epitope within exon 1 of HTT. In embodiments, the antibody binds to an epitope within exon 1 of HTT that does not contain a polyQ tract. In embodiments, the antibody may not bind to proteins derived from CAG repeat-containing genes other than HTT. In other words, the antibodies of the present disclosure may advantageously not exhibit target binding to other proteins expressed from CAG repeat genes (e.g., ataxin, etc.). In embodiments, the antibodies of the present disclosure may bind to high molecular weight / aggregated HTT and soluble forms of HTT / wild-type HTT.

[0086] The HTT can be human HTT or mouse HTT. Preferably, the HTT can be human HTT. HTT can refer to human HTT unless the context indicates otherwise. In other embodiments, for example, when the individual being treated is a non-human mammal, the HTT can be non-human HTT.

[0087] The antibody or fragment thereof may bind to human HTT and may also bind to mouse (murine) HTT antigen. For example, the antibody or fragment thereof may also bind to mouse HTT antigen having the amino acid sequence set forth in SEQ ID NO: 177. Cross-reactivity with mouse HTT antigen may be determined by direct ELISA, for example, by direct ELISA performed as described herein (Example 18, Materials and Methods).

[0088] The present disclosure relates primarily to antibody molecules, whether whole antibodies (e.g., IgG, e.g., IgG4) or antibody fragments (e.g., scFv, Fab, (single domain) dAb). Antibody antigen-binding regions (also called "antigen-binding portions") are provided, as are antibody VH and VL domains. Within the VH and VL domains, complementarity-determining regions (CDRs) are provided, and the CDRs may optionally be provided within different framework regions (FRs) to form the VH or VL domain. An antigen-binding site may consist of an antibody VH domain and / or VL domain.

[0089] Antibodies according to the present disclosure may be provided in isolated form. The term "antibody" includes fragments or derivatives thereof or synthetic antibodies or synthetic antibody fragments.

[0090] The antigen-binding portion can 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 a 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).

[0091] The antibody or fragment thereof may be a monoclonal antibody. A monoclonal antibody (mAb) is a homogeneous population of antibodies that specifically target a single epitope on an antigen.

[0092] Antibody fragments, such as Fab and Fab2 fragments, can also be provided, as can 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 recognized by early protease digestion experiments. Further confirmation was found with the "humanization" of rodent antibodies. Variable domains of rodent origin can be fused to constant domains of human origin so that the resulting antibody retains the antigen specificity of the rodent parent antibody (Morrison et al. (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

[0093] That antigen specificity is conferred by the variable domains and is independent of the constant domains is known from experiments involving the 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 via a flexible oligopeptide (Bird et al. (1988) Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sd.; U.S. Pat. No. 85,5879), and single-domain antibodies (dAbs) containing isolated V domains (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 to be found in Winter & Milstein (1991) Nature 349, 293-299.

[0094] 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, thus allowing the facile production of large amounts of the fragments. Whole antibodies and F(ab')2 fragments are "bivalent." The term "bivalent" means that the antibodies and F(ab')2 fragments have two antigen-binding sites. In contrast, Fab, Fv, ScFv, and dAb fragments are monovalent and have only one antigen-binding site.

[0095] Antibodies according to the present disclosure may be detectably labeled, or at least detectable. For example, antibodies may be labeled with radioactive atoms, colored molecules, fluorescent molecules, or other molecules that can be easily detected. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radioactive labels. The binding moiety (antibody or fragment thereof) may be directly or indirectly labeled with a detectable label. For example, the binding moiety may be an unlabeled antibody that can be detected by another antibody that is itself labeled. Alternatively, the second antibody may be conjugated to biotin, and binding of labeled streptavidin to the biotin is used to indirectly label the first antibody.

[0096] A "fragment" of an antibody may contain any number of residues of a "parent" antibody while retaining target binding ability. A fragment may lack effector function, e.g., may completely fail to bind Fc receptors or may exhibit reduced binding to Fc receptors compared to the parent. Fragments are typically smaller than the parent antibody. A fragment may contain 50%, 60%, 70%, 80%, 90%, 95%, or more of the contiguous or non-contiguous amino acids of the parent antibody. A fragment may contain 50, 100, 150, 200, 250, 300, or more contiguous or non-contiguous amino acids of the parent antibody. A fragment may contain the Fc region or a deletion of the Fc region. A fragment may retain the CDRs and / or variable domains of the parent antibody unchanged. In some embodiments, the fragment is a Fab fragment or a F(ab')2 fragment.

[0097] CDR sequences are described herein using the Kabat definition (Kabat, EA et al., Sequences of Proteins of Immunological Interest.).

[0098] An antibody according to the disclosure can have the CDRs of antibody ATL_5895, 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), iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO:5), vi. LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6).

[0099] The antibodies according to the present disclosure have the V H Sequence and / or V L (a) ATL_5895 V H has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7); and (b) V L has the sequence: QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8).

[0100] An antibody according to the disclosure can have the CDRs of antibody ATL_5901, 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), iv. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9), v. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10), vi. LCDR3 has the amino acid sequence SSYAGSSVV (SEQ ID NO: 11).

[0101] The antibodies according to the present disclosure have the V H Sequence and / or V L (a) ATL_5901 V H has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKATLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 12), and (b) the V L has the sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL (SEQ ID NO: 13).

[0102] An antibody according to the disclosure can have the CDRs of antibody ATL_5567, 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), iv. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); v. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17), vi. LCDR3 has the amino acid sequence SSYAGFSTLV (SEQ ID NO: 18).

[0103] The antibody according to the present disclosure has the V H Sequence and / or V L(a) ATL_5567 V H has the sequence: (a) EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19), and (b) the V L has the sequence: QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGNAPKPLIYEGSKRPSGVSARFSGSSKSGNTASLTISGLQPEDEADYYCSSYAGFSTLVFGGGTKVTVL (SEQ ID NO: 20).

[0104] An antibody of the disclosure can have the CDRs of antibody ATL_5331, 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), iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO:5), vi. LCDR3 has the amino acid sequence GSYAGTNNV (SEQ ID NO: 21).

[0105] The antibodies according to the present disclosure have the V H Sequence and / or V L (a) the VH of ATL_5331 (AC_0737) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19), and (b) the VH of ATL5331L has the sequence: QSALTQPRSVSGSPGQSITISCTGTSSDVGSYNLVSWFQQHPGKAPKLIIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCGSYAGTNNVFGTGTKLTVL (SEQ ID NO: 22).

[0106] An antibody of the disclosure can have the CDRs of ATL_5334, 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), iv. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9), v. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10), vi. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23).

[0107] The antibodies according to the present disclosure have the V H Sequence and / or V L (a) the VH of ATL_5334 (AC_0737) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19), and (b) the VH of ATL5334 L has the sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSKSGSTASLTISGLQAEDEADYYCCSYAGSSVVFGGGTKLTVL (SEQ ID NO: 24).

[0108] An antibody of the disclosure can have the CDRs of ATL_5335, 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), iv. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); v. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17), vi. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25).

[0109] The antibody according to the present disclosure has the V H Sequence and / or V L (a) VH ATL_5335 (AC_0737) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19), and (b) VH ATL5335 (AC_0737) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19). L has the sequence: QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGNAPKPLIYEGSKRPSGVSARFSGSSKSGNTASLTISGLQPEDEADYYCSSYAGFNTLVFGGGTKVTVL (SEQ ID NO: 26).

[0110] An antibody of the disclosure can have the CDRs of ATL_5555, 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), iv. LCDR1 has the amino acid sequence TGTSSDVGSYNLVS (SEQ ID NO: 4); v. LCDR2 has the amino acid sequence EVNKRPS (SEQ ID NO:5), vi. LCDR3 has the amino acid sequence GSYAGTNNV (SEQ ID NO: 21).

[0111] The antibody according to the present disclosure has the V H Sequence and / or V L (a) VH ATL_5555 (AC_1269) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28); and (b) VH ATL_5555 (AC_1269) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28). L has the sequence: QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 29).

[0112] An antibody of the disclosure can have the CDRs of ATL_5556, 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), iv. LCDR1 has the amino acid sequence TGTSSDVGGYKLVS (SEQ ID NO: 9), v. LCDR2 has the amino acid sequence EVSKRPS (SEQ ID NO: 10), vi. LCDR3 has the amino acid sequence CSYAGSSVV (SEQ ID NO: 23).

[0113] The antibody according to the present disclosure has the VH Sequence and / or V L (a) the VH of ATL_5556 (AC_1269) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28); and (b) the VH of ATL_5556 L has the sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSKSGSTASLTISGLQAEDEADYYCCSYAGSSVVFGGGTKLTVL (SEQ ID NO: 24).

[0114] An antibody of the disclosure can have the CDRs of ATL_5557, 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), iv. LCDR1 has the amino acid sequence TGTSSDIGSYNLVS (SEQ ID NO: 16); v. LCDR2 has the amino acid sequence EGSKRPS (SEQ ID NO: 17), vi. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO: 25).

[0115] The antibody according to the present disclosure has the V H Sequence and / or V L(a) VH ATL_5557 (AC_1269) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28); and (b) VH ATL_5557 (AC_1269) has the sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28). L has the sequence: QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYAGFNTLVFGGGTKLTVL (SEQ ID NO: 30).

[0116] Antibodies of the disclosure can have the CDRs of any of antibodies 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 disclosure can have the CDRs of any of antibodies 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 below. H Sequence and / or V L It can have an array: - ATL_6199 VH: [ka] and ATL_6199 VL: QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL, - ATL_6200 VH: [ka] , ATL_6200 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL, - ATL_6202 VH: [ka] , ATL_6202 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL, - ATL_6203 VH: [ka] , ATL_6203 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL, - ATL_6204 VH: [ka] , ATL_6204 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSFAGTANVFGTGTKVTVL, - ATL_6205 VH: [ka] , ATL_6205 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL, - ATL_6194 VH: [ka] , ATL_6194 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL, - ATL_6195 VH: [ka] , ATL_6195 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL, - ATL_6374 VH: [ka] , ATL_6374 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL, - ATL_6375 VH: [ka] , ATL_6375 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL, - ATL_6376 VH: [ka] , ATL_6376 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL, - ATL_6377 VH: [ka] , ATL_6377 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL, - ATL_6378 VH: [ka] , VL of ATL_6378:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL.

[0117] An antibody of the disclosure may have the CDRs of any of antibodies ATL_6183, ATL_6184, ATL_6185, ATL_6186 as provided in Table 1. An antibody according to the disclosure may have the CDRs of any of antibodies ATL_6183, ATL_6184, ATL_6185, ATL_6186 as provided below. HSequence and / or V L It can have an array: - ATL6183 VH: [ka] , ATL6183 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL, - ATL6184 VH: [ka] , ATL6184 VL:QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL, - ATL6185 VH: [ka] , ATL6185 VL:QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL, - ATL6186 VH: [ka] , VL of ATL6186:QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL.

[0118] Antibodies according to the present disclosure may differ in at least one of sequences (i)-(vi). Variants may have one, two, three, four, five, or more (e.g., up to 10, etc.) amino acid substitutions in one or more of sequences (i)-(vi). In embodiments, antibodies according to the present disclosure comprise CDRs having sequences with one, two, or three substitutions compared to sequences (i)-(vi) of any of the antibodies described above. For example, antibodies according to the present disclosure may comprise CDRs having the sequences of any of the antibodies described above, except that one, two, or three of the CDRs contain substitutions, and the total number of substitutions across the CDRs is three or less.

[0119] The VH and VL chain CDRs 1-3 of any of the above antibodies may also be particularly useful in conjunction with several different framework regions. Thus, the light and / or heavy chains having the above CDRs 1-3 may have alternative framework regions. Suitable framework regions are known in the art and are described, for example, in Lefranc & G. Le Franc (2001) "The Immunoglobulin Facts Book," Academic Press.

[0120] As used herein, the antibodies may have VH and / or VL regions comprising amino acid sequences that have a high percentage of sequence identity to the VH and / or VL amino acid sequences set forth above.

[0121] 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% sequence identity, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH region amino acid sequence of any of the antibodies described above (e.g., ATL5895, ATL5901 or 5567, etc.).

[0122] Alternatively, or in addition, an antibody of the present disclosure may have a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL region amino acid sequence of any of the antibodies described above (e.g., ATL5895 or ATL5901 or 5567, etc.).

[0123] The overall percentage identity of a variable region or full-length heavy / light chain sequence can be combined with a particular CDR sequence from the same antibody.

[0124] The antibodies of the present disclosure may contain one or more substitutions within the framework of the VH and / or VL regions. As used herein, a "substitution" refers to the replacement of an amino acid at a particular position with another amino acid relative to the same position in a baseline molecule. In some embodiments, the baseline molecule is an antibody exemplified herein, e.g., ATL_5331, ATL_5334, or ATL_5335.

[0125] In some embodiments, antibodies of the disclosure comprise one or more VH framework substitutions at positions selected from the following group: 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 following VH domain substitutions: D72E, D73E, N76A, T77A, V78L, N82AA, S82BT, S87BA, N83K, D86E, and T87A. In embodiments, the VH framework substitutions are selected from 78L, 83K, and 76A. In embodiments, the VH framework substitutions are (i) 78L and 83K, or (ii) 76A, 78L, and 83K.

[0126] In some embodiments, the VL substitution is at a position selected from the following positions: 8, 19, 36, 42, 46, 47, 60, 69, 75, 80, 104. In some embodiments, the substitution is selected from R8A, I19V, F36Y, N42K, P46L, I47M, A60N, N69A, V75I, P80A, and V104L.

[0127] The sequence and properties of an antibody of the present disclosure may 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 framework, light chain framework, Fc region, and / or hinge region, so long as it binds to the same target, preferably the same epitope, as an antibody of the present disclosure. A reference antibody may be isotype-matched to an antibody of the present disclosure. A reference antibody may bind to the same epitope as an antibody of the present disclosure, or may block, sterically hinder, or otherwise compete for the same epitope as an antibody of the present disclosure. A reference antibody may be known in the art or may have the CDRs and / or variable domains of antibodies in the art, but is otherwise identical to an antibody of the present disclosure. For example, ATL_5059 is a reference antibody (which does not have identical CDR or framework regions to the antibodies of the present disclosure) and is also disclosed as "NI-302.8F1" in US Pat. No. 11,401,325 B2.

[0128] A preferred antibody has one or more residues that differ from a reference antibody capable of binding to the same target and has one or more improved properties compared to the reference antibody. The differences may be in CDR and / or framework residues of the variable domain. In some embodiments, an antibody may differ from a reference antibody in its CDR and optionally bind to the same target at the same or similar epitope. For example, an antibody according to the present invention may exhibit improved binding potency to exon 1 HTT, e.g., improved binding potency to wild-type HTT and / or improved binding potency to mHTT. It is not obvious, if any, to identify residues within an antibody that may improve one or more properties without a posteriori knowledge. This can be achieved by using a "parent" reference antibody and carefully varying individual residues or combinations of residues and analyzing the results. Alternatively, guided design, such as analysis of naturally occurring variants within an antibody family or phage display analysis of antibodies, to identify candidate substitutions can be performed and cross-referenced to produce improved antibodies. Candidate substitutions can be identified from multiple sources and combined for further testing to produce even more advantageous antibodies. The antibodies of the present disclosure were identified independently of any prior art antibodies derived through analysis of the convergence of B-cell repertoires of resilient individuals or derived from such antibodies. Thus, the antibodies of the present disclosure are advantageously derived from naturally occurring protective antibodies, the sequences of which would not have been attainable based on any disclosure in the prior art. For example, the inventors were able to produce antibodies ATL_5331, ATL_5334, and ATL_5335 derived from ATL_5895, ATL_5901, and ATL_5667, respectively, only through the significant research and guided design described herein.

[0129] In embodiments, an isolated antibody or antibody fragment thereof that specifically binds to a huntingtin (HTT) protein or a 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, and the position numbering is Kabat; ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15) or an amino acid sequence comprising one or two amino acid substitutions compared to RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), and optionally optionally, the substitution is selected from 64, 54 or 53, optionally the substitution is selected from Q64K, D53E and G54A, where the position numbering is according to Kabat; and iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3) or a sequence comprising 1, 2, 3 or 4 substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3), optionally the substitution is selected from 95, 97, 100A, 100B, 100C, optionally the position is selected from Y97F, P95S, Y100AG, G100BL and L100C, where the position numbering is according to Kabat.

[0130] 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 TGTSSDVGSYNLVS (SEQ ID NO: 4), TGTSSDVGGYKLVS (SEQ ID NO: 9), TGTSSDIGSYNLVS (SEQ ID NO: 16); v. LCDR2 has an amino acid sequence selected from EVNKRPS (SEQ ID NO: 5), EVSKRPS (SEQ ID NO: 10), EGSKRPS (SEQ ID NO: 17); and vi. LCDR3 is GSYAGTNNV (SEQ ID NO: 21), an amino acid sequence comprising one, two or three amino acid substitutions compared to GSYAGTNNV (SEQ ID NO: 21), optionally wherein the substitutions are selected from 92, 95, 89 and 91, optionally wherein the substitutions are A92G, N95 and Y91F, an amino acid sequence selected from the group consisting of: CSYAGSSVV (SEQ ID NO: 23), an amino acid sequence comprising one or two amino acid substitutions compared to CSYAGSSVV (SEQ ID NO: 23), optionally the amino acid substitution is selected from 89, 95, and optionally the substitution is selected from C89S, N95A; and the amino acid sequence 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 the amino acid substitution is at position N95 and / or T95, and optionally the substitution is selected from N95S and / or T95A, wherein the position numbering is according to Kabat.

[0131] 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 that comprise 1 to 10 substitutions compared to the following CDRs: i. 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 the sequence of HCDR1 comprises one substitution compared to KAWMN (SEQ ID NO: 14) or KAWMS (SEQ ID NO: 1); ii. an HCDR2 having the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2), RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27), or Optionally, the sequence of HCDR2 comprises 1, 2, 3, 4, 5, or 6 substitutions compared to RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), RIKSGIDAGTTDYAAPVKG (SEQ ID NO: 2), or RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27); iii. an HCDR3 having the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3), optionally wherein the sequence of HCDR3 comprises 1, 2, 3 or 4 substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3); iv. LCDR1 having the 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); v. LCDR2 having the amino acid sequence EVNKRPS (SEQ ID NO: 5), EVSKRPS (SEQ ID NO: 10), or EGSKRPS (SEQ ID NO: 17); vi. An LCDR3 having the amino acid sequence: 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).

[0132] In embodiments, the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, where 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, where 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 another embodiment, 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).

[0133] In embodiments, the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, where i. HCDR1 has the amino acid sequence KAWMN (SEQ ID NO: 14), ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVQG (SEQ ID NO: 15), 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, where 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 another embodiment, 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 another embodiment, 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 another embodiment, 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).

[0134] In embodiments, the antibody comprises a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2, and HCDR3, where i. HCDR1 has the amino acid sequence KAWMS (SEQ ID NO: 1), ii. HCDR2 has the amino acid sequence RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27), 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, where 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 GSYAGTNNV (SEQ ID NO: 21). In another embodiment, 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 another embodiment, 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).

[0135] In embodiments, the antibody 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 RIKSGIDGGTTDYAAPVKG (SEQ ID NO: 27); and iii. HCDR3 has the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3), or the amino acid sequence SPYYYYYGLDV (SEQ ID NO: 157), or the amino acid sequence PPFYYYYGLDV (SEQ ID NO: 158), or the amino acid sequence 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); and 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), and 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), and iii. LCDR3 has the amino acid sequence SSYAGFNTLV (SEQ ID NO:25).

[0136] 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_0005331 VH]: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-CDRH1-WVRQAPGKGLEWVG (SEQ ID NO: 99)-CDRH2-RFTISRDDSKNTVYLQMNSLNTEDTAVYYCIP (SEQ ID NO: 100)-CDRH3-WGQGTTVTVSS (SEQ ID NO: 101); (b) the framework sequence of [ATL_0005334 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) ATL_0006199 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCSP (SEQ ID NO: 169)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101); (b) ATL_0006200 Framework sequence of VH (and ATL_0006374): EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVP (SEQ ID NO: 170)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), (c) ATL_0006202 Framework sequence of VH: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCSP (SEQ ID NO: 171)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), (d) ATL_0006203 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCIP (SEQ ID NO: 172)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), (e) ATL_0006205 Framework sequences of VH (and ATL_0006375, ATL_0006376, ATL_0006377 and ATL_0006378): EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCVP (SEQ ID NO: 173)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101).The isolated antibody or fragment thereof may comprise one or more framework substitutions in the VH domain (e.g., compared to the framework sequences above). The one or more framework substitutions may be at a position selected from 72, 73, 76, 77, 78, 82A, 82B, 83, 86, and 87. The one or more framework substitutions may be at a position selected from 72, 73, 76, 77, 78, 82A, 82B, 83, 86, 87, 91, and 93. The one or more framework substitutions in the VH domain may be selected from the group consisting of: E at position 72 (e.g., D72E), E at position 73 (e.g., D73E), A at position 76 (e.g., N76A), A at position 77 (e.g., T77A), L at position 78 (e.g., V78L), A at position 82A (e.g., N82AA), T at position 82B (e.g., S82BT), A at position 82B (e.g., S87BA), K at position 83 (e.g., N83K), E at position 86 (e.g., D86E), A at position 87 (e.g., T87A), W at position 91 (Y91W), and M, S, or V at position 93 (I93M, I93S, I93V), where position numbering is according to Kabat. Such mutations in the framework regions may advantageously eliminate trends, improve the stability, and reduce immunogenicity of the resulting antibody. For example, mutations D72E and / or D73E may reduce the risk of isomerization, N76A and / or T77A may reduce the risk of deamination, D86E and / or T87A may reduce the risk of isomerization, and mutations N82AA, S82BT, N83K, and / or S87BA may reduce the risk of deamination. In embodiments, the framework substitutions are selected from 78L, 83K, and 76A; optionally, the framework substitutions are (i) 78L and 83K, or (ii) 76A, 78L, and 83K.

[0137] In embodiments, the VL domain is a human VL domain. In embodiments, the antibody or fragment thereof has a VL domain framework sequence selected from the following: (a) the framework sequence of [ATL_0005331 VL]: QSALTQPRSVSGSPGQSITISC (SEQ ID NO: 102)-CDRL1-WFQQHPGKAPKLIIY (SEQ ID NO: 103)-CDRL2-GVPDRFSGSKSGNTASLTVSGLQAEDEADYYC (SEQ ID NO: 104)-CDRL3-FGTGTKLTVL (SEQ ID NO: 105); and (c) the framework sequence of [ATL_0005335 VL]: QSALTQPASVSGSPGQSITISC (SEQ ID NO: 106)-CDRL1-WYQQHPGRAPKLMIY (SEQ ID NO: 107)-CDRL2-GVSSRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 108)-CDRL3-FGGGTKLTVL (SEQ ID NO: 109). In embodiments, the isolated antibody or fragment thereof comprises one or more framework substitutions in the VL domain (e.g., compared to the framework sequences above). The one or more framework mutations may be at a position selected from 8, 19, 36, 42, 46, 47, 60, 69, 75, 80, 104. The one or more framework substitutions in the VL domain may be selected from the group consisting of: position 8: R (e.g., R8A), position 19: V (e.g., I19V), position 36: Y (e.g., F36Y), position 42: K (e.g., A60N), position 69: L (e.g., P46L), position N69A: M (e.g., I47M or L47M), position 60: N (e.g., N42K), position 46: A (e.g., 47), position 75: I (e.g., V75I), position 80: A (e.g., P80A), and position 104: V or L (e.g., L104V or V104L), where position numbering is according to Kabat.The one or more framework mutations can be at a position selected from the following: (i) 8 (optionally, the substituted amino acid is R) in antibody ATL_0005334, antibodies derived therefrom, such as ATL_0005586, ATL_0005900, ATL_0005556, ATL_0005901, or antibodies comprising the VL sequence of any of ATL_0005586, ATL_0005900, ATL_0005901, ATL_0005556 (e.g., SEQ ID NOs: 13, 24); (ii) 8 (optionally, the substituted amino acid is R) in antibody ATL_0005331 or antibodies derived therefrom. antibodies, such as ATL_0005577, ATL_0005891, ATL_0005890, ATL_0005559, ATL_0005563, ATL_0005896, ATL_0005894, ATL_0005895, ATL_0005555 or ATL_0005577, ATL_0005891, ATL_0005890, ATL_0005559, ATL_0005563, ATL_0005896, ATL_0005894, ATL_0005895, ATL_0005555 (e.g., SEQ ID NOs: 22, 32, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 19 (optionally, the substituted amino acid is V), 36 (optionally, the substituted amino acid is Y), 47 (optionally, the substituted amino acid is M), 69 (optionally, the substituted amino acid is A), 75 (optionally, the substituted amino acid is I), 104 (optionally, the substituted amino acid is V) in an antibody comprising any of the VL sequences of ATL_0005335 or antibodies derived therefrom, such as ATL_0005572, ATL_0005567, ATL_0005557 or ATL_0005338 42 (optionally, the substituted amino acid is R), 46 (optionally, the substituted amino acid is K), 47 (optionally, the substituted amino acid is M), 60 (optionally, the substituted amino acid is N), 69 (optionally, the substituted amino acid is A), 80 (optionally, the substituted amino acid is A), 104 (optionally, the substituted amino acid is L) in an antibody comprising a VL sequence comprising any of ATL_0005572, ATL_0005567, ATL_0005557 (e.g., SEQ ID NOs: 30, 26, 20, 39).Such mutations in framework regions may advantageously eliminate the tendency, improve the stability and reduce immunogenicity of the resulting antibody. For example, substitution N69A (or 70A) may reduce the risk of deamination.

[0138] In embodiments, HCDR1, HCDR2 and HCDR3 of the VH domain are in a germline framework, hi embodiments, LCDR1, LCDR2 and LCDR3 of the VL domain are in a germline framework.

[0139] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_5895 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_5895 VL). In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKATLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 12) (ATL_5901 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL (SEQ ID NO: 13) (ATL_5901 VL). In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5567 VH).In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGNAPKPLIYEGSKRPSGVSARFSGSSKSGNTASLTISGLQPEDEADYYCSSYAGFSTLVFGGGTKVTVL (SEQ ID NO: 20) (ATL_5567 VL).

[0140] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5331 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSITISCTGTSSDVGSYNLVSWFQQHPGKAPKLIIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCGSYAGTNNVFGTGTKLTVL (SEQ ID NO: 22) (ATL_5331 VL).

[0141] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5334 VH). In some such embodiments, the light chain variable domain sequence comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCCSYAGSSVVFGGGTKLTVL (SEQ ID NO: 24) (ATL_5334 VL).

[0142] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5335 VH). In some such embodiments, the light chain variable domain sequence is QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGNAPKPLIYEGSKRPSGVSARFSGSSKSGNTASLTISGLQPEDEADYYCSSYAGFNTLVFGGGTKVTVL (SEQ ID NO: 26) (ATL_5335 VL).

[0143] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28) (ATL_5555 VH). In some such embodiments, the light chain variable domain sequence is QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 29) (ATL_5555 VL).

[0144] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28) (ATL_5556 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCCSYAGSSVVFGGGTKLTVL (SEQ ID NO: 24) (ATL_5556 VL).

[0145] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28) (ATL_5557 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYAGFNTLVFGGGTKLTVL (SEQ ID NO: 30) (ATL_5557 VL).

[0146] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 31) (ATL_5577 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSITISCTGTSSDVGSYNLVSWFQQHPGKAPKLIIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCGSYAGTNNVFGTGTKLTVL (SEQ ID NO: 22) (ATL_5577 VL). In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 31) (ATL_5891 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIYEVNKRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 32) (ATL_5891 VL).

[0147] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 31) (ATL_5586 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL (SEQ ID NO: 13) (ATL_5586 VL).

[0148] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 31) (ATL_5890 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 29) (ATL_5890 VL).

[0149] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5559 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSITISCTGTSSDVGSYNLVSWFQQHPGKAPKLIIYEVNKRPSGVPDRFSGSKSGATASLTVSGLQAEDEADYYCGSYAGTNNVFGTGTKLTVL (SEQ ID NO: 42) (ATL_5559 VL). In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5572 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDIGSYNLVSWYQQHPGKAPKPLIYEGSKRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYAGFNTLVFGGGTKLTVL (SEQ ID NO: 39) (ATL_5572VL).

[0150] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 19) (ATL_5563 VH).

[0151] In embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIYEVNKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 32) (ATL_5563 VL).

[0152] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_5896 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 40) (ATL_5896 VL).

[0153] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_5894 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 41) (ATL_5894 VL).

[0154] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 28) (ATL_5900 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL (SEQ ID NO: 13) (ATL_5900 VL).

[0155] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMNWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVQGRFTISRDDSKNTVYLQMNSLNTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 31) (ATL_5891 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIYEVNKRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCGSYAGTNNVFGTGTKVTVL (SEQ ID NO: 32) (ATL_5891 VL).

[0156] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCSPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 144) (ATL_6199 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6199 VL).

[0157] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6200 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6002 VL).

[0158] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCSPPPFYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 146) (ATL_6202 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6202 VL).

[0159] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 147) (ATL_6203 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6203 VL).

[0160] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_6204 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSFAGTANVFGTGTKVTVL (SEQ ID NO: 155) (ATL_6204 VL).

[0161] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 148) (ATL_6205 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 156) (ATL_6205 VL).

[0162] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTPPPYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 149) (ATL_6183 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6183 VL).

[0163] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKATLYLQMNSLKTEDTAVYYCTPPPYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 150) (ATL_6184 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL (SEQ ID NO: 13) (ATL_6184 VL).

[0164] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTPPPYYYYGLNVWGQGTTVTVSS (SEQ ID NO: 151) (ATL_6185 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6185 VL).

[0165] In embodiments, the heavy chain variable domain comprises the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKATLYLQMNSLKTEDTAVYYCTPPPYYYYGLNVWGQGTTVTVSS (SEQ ID NO: 152) (ATL_6186 VH). In some such embodiments, the light chain variable domain comprises the amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYKLVSWYQQHPGRAPKLMIYEVSKRPSGVSSRFSGSSKSGSTASLTISGLQAEDEADYYCSSYAGSSVVFGGGTKLTVL (SEQ ID NO: 13) (ATL_6186 VL).

[0166] 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 one of the above heavy chain variable domains (e.g., SEQ ID NOs: 7, 12, 19, 28, 31). In embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% sequence identity to any one 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).

[0167] In some embodiments, the antibodies of the invention have improved binding potency compared to a reference antibody. The improved binding potency can be achieved by modifying one or more residues in the CDRs or V H or V L The binding efficacy can be determined by the half-maximal effective concentration (EC 50 ) value or the concentration required to obtain 50% binding. Binding efficacy can be measured using ELISA-based assays known in the art, such as the HTT sandwich ELISA.

[0168] In some embodiments, the isolated antibody or fragment thereof binds to mHTT and / or aggregated HTT protein (or preferably a fragment thereof that includes exon 1) as determined by immunoprecipitation (e.g., immunoprecipitation of mHTT and / or aggregated HTT using an antibody or fragment thereof of the present disclosure).

[0169] In some embodiments, an antibody or fragment thereof according to the present disclosure can cross the blood-brain barrier. In some embodiments, the antibody is a bispecific antibody. For example, an antibody according to the present disclosure may have one scFv chain that binds to a receptor in the brain, such as the transferrin receptor (Yu et al., Sci Transl Med. 2014 Nov 5; 6 (261): 261ral54.), and one scFv chain that binds to HTT as described herein. In some embodiments, an antibody according to the present disclosure comprises an antibody or fragment thereof (e.g., an antibody, scFv, sdAb, etc.) that binds to HTT as described herein, and an additional binding moiety that binds to another target. The other target may be a receptor in the brain, such as a transduction receptor. The additional binding moiety may be an antibody, scFv, nanobody, or aptamer. The two binding moieties of such a bispecific molecule may form a fusion protein.

[0170] Also described herein are single domain antibodies (sdAbs), also known as nanobodies, comprising the heavy chain CDR and / or VH sequence of any of the antibodies described herein. Accordingly, also described herein are antibodies or fusion molecules comprising a nanobody that binds to HTT as described herein and a nanobody that binds to a receptor in the brain. Also described herein are antibodies that are fusion molecules comprising an scFV chain or nanobody that binds to HTT as described herein and an aptamer that binds to a receptor in the brain.

[0171] Isolated nucleic acids encoding the antibodies, antigen-binding fragments, or polypeptides described herein are provided. Also provided are vectors containing the nucleic acids described herein and host cells containing the vectors. For example, the host cells can be eukaryotic or mammalian, such as Chinese hamster ovary (CHO) cells, or prokaryotic, such as Escherichia coli (E. coli). In some embodiments, the vector is a viral vector, such as a bacteriophage.

[0172] Further provided is a method of making an antibody or antigen-binding fragment or polypeptide described herein, comprising culturing a host cell described herein under conditions suitable for expression of 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 comprising formulating the antibody or antibody fragment into a composition comprising at least one additional component.

[0173] The antibodies and fragments thereof described herein can be used therapeutically.

[0174] 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. Therapeutic use can be in humans or animals (veterinary use).

[0175] Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by several routes, including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, oral, and nasal. Medicaments and compositions may be formulated for injection.

[0176] Pharmaceutical compositions can be prepared using pharmaceutically acceptable "carriers" composed of materials considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., physiologically tolerated, and typically do not cause allergic or similar adverse reactions, such as stomach upset, when administered to humans. In some embodiments, the term refers to molecular entities and compositions that have been recognized by a U.S. federal or state regulatory agency as being GRAS-listed under Sections 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, subject to prior review and approval by the FDA or similar listing, the U.S. Pharmacopeia, or another generally recognized pharmacopeia, 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 for formulating pharmaceutical compositions using such carriers.

[0177] The pharmaceutical compositions provided herein may contain one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonicity agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., the anti-CFH antibody used in the composition. Thus, those skilled in the art will understand that a composition is provided in which there is no incompatibility between any of the components of the dosage form. The excipient may be selected from the group consisting of a buffer, a solubilizer, an isotonicity agent, a chelating agent, an antioxidant, an antimicrobial agent, and a preservative.

[0178] Administration is preferably in a "therapeutically effective amount," which is 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 condition being treated. Prescribing treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other doctors, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0179] Conditions treatable according to the present disclosure include any condition in which HTT plays a role, including neurodegenerative disorders, particularly conditions characterized by the pathological accumulation of abnormal protein aggregates, such as mHTT aggregates, in the brain. Conditions treatable according to the present disclosure include any polyQ (polyglutamine)-associated disease (see, e.g., Cell Transplant. 2014; 23 (4-5): 441-58). PolyQ diseases are neurodegenerative disorders caused by expanded CAG repeats in certain proteins. These include spinocerebellar ataxia type 6 (SCA) types 1, 2, 6, 7, and 17, Machado-Joseph disease (SCA3), Huntington's disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), and spinal-bulbar muscular atrophy, X-linked type 1 (SMAX1). The antibodies of the present disclosure were originally identified through analysis of resilient Alzheimer's disease patients and have shown relevance beyond HD to other aggregation-related neurodegenerative diseases or disorders, including one or more of the following: Huntington's disease, Alzheimer's disease (AD), frontotemporal dementia, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), prion diseases, Lewy body disease, spinal muscular atrophy (SMA), motor neuron disease (MND), progressive supranuclear palsy (PSP), spinocerebellar ataxia (SCA) types 1, 2, 6, 7, and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy, X-linked type 1 (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease), and DNAJB6 myopathy.

[0180] The antibodies of the present disclosure may be used in treatment with an additional therapeutic agent. As used herein, an "additional therapeutic agent" is an additional compound, protein, vector, antibody, cell, or entity that has a therapeutic effect. The antibody may be co-administered with the additional therapeutic agent. The antibody may be co-formulated with the additional therapeutic agent. The antibody may be administered sequentially, before or after the additional therapeutic agent.

[0181] 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 disorder characterized by the presence of mutated or aggregated HTT protein in a patient. The presence of mutant and / or aggregated HTT indicates disease progression. HTT levels can be quantified in cerebrospinal fluid or blood and in samples derived from the patient. The levels of mutant and / or aggregated protein can be quantified using any technique known in the art. Various assays are available, including ELISA, flow cytometry, and Western blot. Thus, also described herein are methods for detecting the presence and / or amount of mutant or aggregated HTT protein in a sample (e.g., a sample obtained from a patient diagnosed with or suspected of having a disease or disorder characterized by the presence of mutant or aggregated HTT protein), comprising using the antibodies or fragments thereof described herein (e.g., to label, isolate, etc., mutant or aggregated HTT present in the sample).

[0182] The antibodies described herein can be used as biomarkers that indicate whether a subject has or is likely to develop a disease or disorder characterized by the presence of mutant or aggregated HTT protein. For example, a method for diagnosing a disease or disorder characterized by the presence of mutant or aggregated HTT protein in a patient can include obtaining BCR sequence data from the subject and using the sequence data to determine whether the subject's BCR repertoire includes one or more antibodies that can bind to HTT (e.g., an antibody described herein, e.g., 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 of the antibodies or antibody fragments described herein), and subjects whose BCR repertoire includes one or more antibodies that can bind to HTT are likely to have or are at risk of developing a disease or disorder characterized by the presence of mutant or aggregated HTT.

[0183] The antibodies described herein can be used as biomarkers that indicate a subject is likely to respond to treatment using the antibodies or antibody fragments described herein. A method for determining whether a subject is likely to respond to treatment with the antibodies or antibody fragments thereof described herein includes obtaining BCR sequence data from the subject and using the sequence data to determine whether the subject's BCR repertoire includes one or more antibodies that are likely to bind to HTT (e.g., an antibody described herein, e.g., 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 particular antibody or antibody fragment described herein), wherein subjects whose BCR repertoire does not include one or more antibodies that are likely to bind to HTT are likely to respond to treatment with the antibodies or antibody fragments thereof described herein. Thus, described herein is a method of treating a subject diagnosed as having or likely to have a disease or disorder associated with HTT and / or polyQ aggregation (e.g., a neurodegenerative disorder), the method comprising obtaining BCR sequence data from the subject, using the sequence data to determine whether the subject's BCR repertoire includes one or more antibodies that are likely to bind to HTT, and administering a therapeutically effective amount of an antibody or antibody fragment thereof described herein to a subject whose BCR repertoire does not include one or more antibodies that are likely to bind to HTT.

[0184] Some methods of the present disclosure involve a sample containing cells. The sample may be a culture of cells grown in vitro. For example, the culture may include cells or a suspension of cells cultured in a culture plate or dish. Methods according to the present disclosure may be performed or products may exist in vitro, ex vivo, or in vivo. The term "in vitro" is intended to encompass experiments using materials, biological substances, cells, and / or tissues in laboratory conditions or in culture, while the term "in vivo" is intended to encompass experiments and procedures using intact multicellular organisms. "Ex vivo" refers to something that exists or originates outside of an organism, e.g., outside the human or animal body, which may be present on tissues (e.g., whole organs) or on cells removed from an organism.

[0185] According to some aspects of the present disclosure, a kit of parts is provided comprising an antibody according to the invention, hi some embodiments, the kit comprises an antibody according to the invention and one or more of: reagents for use in immunochemistry; an antibody immobilized on a solid support; a means for labeling the antibody; a means for linking the antibody to a cytotoxic moiety; and an additional therapeutic agent.

[0186] The percentage of sequence identity (%) is defined as the percentage of amino acid residues in the candidate sequence that are identical to the residues in the comparison sequence, after aligning the sequences to achieve maximum sequence identity and introducing gaps as necessary, without considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of each sequence. If the aligned sequences are of different lengths, the sequence identity of the shorter comparison sequence can be determined over the entire length of the longer given sequence, or if 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 the algorithm GAP (Wisconsin GCG Package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters can be used, with a gap creation penalty of 12 and a gap extension penalty of 4. While the use of GAP may be preferred, other algorithms may be used, such as BLAST (Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448)), SSEARCH (Smith and Waterman (1981) J. Mol. Biol. 147: 195-197; Pearson Genomics. 1991 Nov; 11 (3): 635-50), HMMER3 (Johnson LS et al BMC Bioinformatics. 2010 Aug 18; 11 (): 431) or Altschul et al. (1990), supra (generally employing default parameters (e.g., Pearson Curr Prot Bioinformatics (2013) Chapter 3 Univ. 3.1) doi: 10.1002 / 0471250953.bi0301s42) and the TBLASTN program.In particular, the psi-Blast algorithm may be used (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402). Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Worcester, Massachusetts, USA). Sequence comparison is preferably performed over the entire length of the relevant sequences being compared. Features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings may be expressed in specific forms thereof, or in terms of means for performing a disclosed function or methods or processes for obtaining a disclosed result, and may, as appropriate, be utilized separately or in any combination of such features to realize the invention in various of its forms.

[0187] While 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 given this disclosure. Accordingly, the exemplary embodiments of the present invention set forth above are considered to be illustrative and not limiting. Various modifications to the described embodiments can be made without departing from the spirit and scope of the invention.

[0188] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0189] Throughout this specification, including the claims that follow, unless the context requires otherwise, the words "comprise" and "comprises," and variations such as "comprises," "including," and "comprising," are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may 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 the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that the particular value forms another embodiment by use of the antecedent "about." The term "about" in reference to numerical values ​​is optional and means, for example, + / - 10%.

[0190] All references cited herein are incorporated by reference in their entirety. For standard molecular biology techniques, see Sambrook, J., Russell, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0191] array

[0192] [Table 1]

[0193] [Table 2]

[0194] Table 3

[0195] Table 4

[0196] Table 5

[0197] Table 6

[0198] Table 7

[0199] Table 8

[0200] Table 9

[0201] Table 10

[0202] Table 11

[0203] Table 12

[0204] Table 13 [Example]

[0205] Example The following examples demonstrate the process of identifying and characterizing exemplary HTT antibodies of the present disclosure.

[0206] Materials and Methods Capillary isoelectric focusing Charge variant analysis was performed on the lead antibody by preparing a master mix for diluting the antibody sample and running it on a cIEF cartridge on a Maurice instrument (Protein Simple). The master mix had final concentrations of 0.35% methylcellulose, 4% pharmalyte 3-10, 10 mM arginine, and 0.01% pI markers 4.09 and 9.99. Samples were diluted to 0.15-0.25 mg / ml in the master mix and run at 1500 volts for 1 minute, followed by a 4.5-minute run at 3000 volts. System suitability standards (Protein Simples) were also run at the beginning and end of the run. Data generated for the same sample under different stress conditions were overlaid to compare charge species profiles.

[0207] 5 FT cycles A 5 mg / ml mAb sample was freeze-thawed from -80C storage to room temperature (RT) for five cycles within 8 hours. The final sample after five freeze-thaw cycles was diluted to 0.7 mg / ml for purity analysis by SEC-HPLC.

[0208] SEC-HPLC Antibody samples were diluted to 0.7 mg / ml in 20 mM histidine acetate, 150 mM NaCl, pH 5.5, and run on a Zorbax GF-250 SEC-HPLC column (Agilent) in a Vanquish Flex (Thermo). Samples were separated by size 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. Chromeleon software (Thermo) was used to integrate chromatograms.

[0209] Recombinant HTT exon-1 binding ELISA Indirect ELISA was performed using two different constructs of recombinant HTT exon-1 with His and GST tags (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 His tags (WT human HTT exon 1 25Q His and WT human HTT exon 1 48Q His). Each construct or an unrelated protein, lysozyme, was coated onto Nunc Maxsorp plates at 5 μg / ml in PBS and incubated overnight at 4°C. The plates were blocked with PBS + 2% nonfat dry milk for 2 hours at room temperature. The buffer was discarded, and the plates were washed three times with PBS + 0.1% Tween-20. Test or control antibodies were added to the wells at 100 μg / ml in PBS + 2% nonfat dry milk 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. HRP-conjugated secondary antibody in PBS + 2% nonfat dry milk 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 for 5-10 minutes at room temperature, then stopped with 0.2M NaOH. The plate was read to measure absorbance at 450 nm.

[0210] Thermal shift assay Protein thermal shift measurements were performed using a QuantiStudio 5 real-time qPCR system (ThermoFisher). Antibodies were diluted 0.1-0.25 with 10x Sypro orange protein gel stain (Thermo #S6651). Samples were loaded into the qPCR system in a 384-well Microamp plate. Samples were run over a temperature range of 25-95°C at 2-minute intervals. Protein melting curves were analyzed using Protein Thermal Shift software to determine Tm values ​​related to antibody stability.

[0211] HTT sandwich ELISA To evaluate the binding potency of antibodies to HTT protein, antibodies were evaluated in a sandwich ELISA format for binding to HTT exon 1 with 25Q repeats or 48Q repeats (see "Sequences" for sequences), and 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 per well of a 96-well plate and left at 4°C overnight. Plates were washed with PBS / 0.1% Tween. Plates were blocked with 50 μl / well of blocking buffer (1% BSA / PBS). Plates were washed with PBS / 0.1% Tween. 50 μl of diluted antigen of interest (HTT exon 1 48Q GST or HTT exon 1 25Q GST) and lysozyme (negative antigen control) were added to the plate at 0.04 μg / ml. The plate was incubated at room temperature for 1 hour on a plate shaker (300 rpm). The plate was washed with PBS / 0.1% Tween. Eight-point, 3-fold serial dilutions of the test antibody (typically starting at 400 nM) were added to the plate. Another set of wells received 50 μl / well of buffer only (blank control). The plate was incubated at room temperature for 1 hour on a plate shaker (300 rpm). The plate was washed with PBS / 0.1% Tween. 50 μl per well of anti-human IgG HRP (80 ng / ml, Jackson ImmunoResearch, #109-035-097, lot: 160716) was added. Plates were washed with PBS / 0.1% Tween and 50 μl of TMB solution (Lifetechnology, #002023) was added. Incubation was continued for 6 minutes at room temperature in the dark. 50 μl of stop solution was added to the plate (Fither Chemical, #12933634). Absorbance was read at 450 nm on a CLARIO Star. Background (average from wells without test antibody) was subtracted from all values. Data analysis was performed using EC50 (half-maximal concentration) values ​​calculated using unconstrained, nonlinear, four-parameter curve fitting.

[0212] In vivo PK studies To determine serum PK of the lead antibody, in vivo PK experiments were performed using ATL_0005335 (the parent antibody of ATL_0005567). Female 6-8 week-old C57BL6J mice received IP injections of ATL_0005335 at 10 and 20 mg / kg, and serum was collected at 1, 4, 8, 24, 72, and 144 hours post-dose. Antibody levels were assessed by ELISA in serum samples.

[0213] HTT immunoprecipitation from U-2 OS cell lysates Immunoprecipitation was performed using Protein G-coated Dynabeads™ and a magnetic rack (heat sealer, 10014D). After incubation with the antibody, the beads were washed using a magnetic rack and incubated with U-2 OS cell lysate (from parental or HTT 110CAG-expressing versions). The beads were washed again, and then the captured proteins were eluted and analyzed by automated Western blot (Bio-techne, Jess). Detection was performed using anti-HTT antibody 1C2 (Merck / Millipore MAB1574).

[0214] HTT immunoprecipitation from R6 / 2 mouse brain Immunoprecipitation was performed using Protein G-coated Dynabeads™ and a magnetic rack (Thermofisher, 10014D). After incubation with the antibody, the beads were washed using a magnetic rack and incubated with brain homogenates from R6 / 2 mice or non-transgenic (non-Tg) littermates. The beads were washed again, and then the captured proteins were eluted and analyzed by Western blot. Detection was performed with anti-HTT antibodies MW8 (Merck / Millipore MABN2529) and 1C2 (MAB1574 Sigma-Aldrich). High molecular weight HTT material was successfully immunoprecipitated by ATL_0005895, ATL_0005901, and ATL_0005567 (Figure 17B) and ATL_0005335 (data not shown).

[0215] HTT immunoprecipitation from human Huntington's disease brains Brain homogenates were prepared from superior temporal gyrus tissue from postmortem Huntington's disease patients. Homogenates were prepared in brain lysis buffer (BLB) containing benzonase and protease inhibitors (10 mM Tris-HCl pH 7.4, 0.8 M NaCl, 1 mM EDTA, 10% sucrose). The homogenates were centrifuged at 2,700 × g, and the supernatants were used for IP. Protein G-coated Dynabeads were coupled to isotype control antibodies or ATL_0005895 and then IP was performed with brain homogenates in brain lysis buffer overnight on a rotating wheel at 4 °C. The next day, the beads were washed with BLB, then heat-denatured in Western blot sample buffer and analyzed by Western blot. Detection was performed using anti-HTT antibodies HD1 and MW1 (MABN2427, Millipore).

[0216] Phagocytosis assay To monitor phagocytic activity in cultures, Q48HTT-coated latex beads (Invitrogen, 11564067) were labeled with pH-sensitive pHrodo™ Red Succinimidyl Ester (Thermo Fisher, P36600). Induced pluripotent stem cell (iPSC)-derived microglia (Fujifilm, C1110) were seeded at 23,000 cells / well in 96-well plates and rested for 3 days with a 50% medium change on day 3. On day 4, 0.5% latex bead solutions were incubated with antibodies at concentrations of 1, 2.1, 4.2, 8.3, 16.7, 33.3, 133.3, 266.7, and 333.3 (nM) and added to the microglia at a 1 / 80 dilution. Phagocytosis was monitored over 4 hours using an Incucyte (Sartorius) microscope.

[0217] Phagocytosis of the beads induces a red fluorescent signal in response to the low pH intracellular environment. The change in the total area of ​​the red fluorescent signal over time indicates the rate of phagocytosis and the amount of bait taken up. To assess the effect of ATL_0005895 (also referred to herein as ATLX_1095) on phagocytosis, the bead-48QHtt-pHrodo™ Red complex was incubated with either ATL_0005895 or a human IgG1 isotype control antibody for 1 hour before exposing cells to the antibody-treated bead-48QHtt-pHrodo™ Red complex.

[0218] In vivo PK assay To characterize the serum PK of select HTT antibodies, in vivo PK experiments were performed using ATL_5567 and ATL_5901 at 10 mg / kg. Male 6-8 week-old C57BL6J mice were administered 10 mg / kg of the relevant antibody by intraperitoneal injection (IP), and serum was collected at 1, 4, 8, 24, 72, and 144 hours post-dose. Antibody levels were assessed by ELISA of serum samples. ATL_5895 was tested at 1, 10, and 60 mg / kg to determine serum and CSF PK. Male 6-8 week-old C57BL6J mice received 10 mg / kg of the relevant antibody by IP injection. Serum was collected at 1, 4, 8, 24, 72, and 144 hours post-dose. CSF was collected at 4 and 144 hours post-dose.

[0219] In vivo PD assay To evaluate the effect of ATL_5895 on HTT aggregate burden in R6 / 1 mice, in vivo PD experiments were performed using 60 mg / kg ATL_5895. Mice used were mixed-sex R6 / 1 mice (Jackson Laboratory stock number 006471). They were 5 weeks old at the start of the study and treated for up to 12 weeks (17 weeks of age). Non-Tg littermates served as wild-type controls. Mice were administered 60 mg / kg ATL_5895 or vehicle control (histidine acetate buffer) via IP once a week. Tissue samples were collected after 0, 4, 8, and 12 weeks of treatment. Samples for HTT analysis were snap-frozen in liquid nitrogen and stored at -80°C until analysis. For analysis, samples were prepared as lysates in MSD lysis buffer supplemented with NaF, PMSF, and protease inhibitor cocktails 1 (Mini, EDTA-free, catalog no. 04693159001, Roche), 2 (Sigma, catalog no. P5726), and 3 (Sigma, catalog no. P0044). Samples from the striatum and cortex were then analyzed for aggregated HTT by a Mesoscale Discovery (MSD) assay using the 4C9 / MW8 antibody pair. Soluble mutant HTT levels were determined by an MSD assay using the 2B7 / MW1 antibody pair. Endogenous mouse HTT levels were determined by an MSD assay 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.

[0220] phage display Phage libraries of scFv sequences derived from ATL_5895 were generated. These libraries contained: (1) VH sequences either soft-randomized at the CDR3H (I105-V117, IMGT numbering corresponds to 93-102 Kabat) or hard-randomized at Y103 (Y91), I105 (I93), P106 (P94 Kabat), G114 (G100B), and L115 (L100C) (IMGT numbering, Kabat numbers in parentheses), and (2) VL or CDR3L soft-randomized variants of ATL_5895 (randomized G105-V117, IMGT numbering corresponds to 89-97 Kabat). Soft randomization was performed using degenerate oligonucleotides synthesized with a 70-10-10-10 mixture of nucleotide bases, with an excess of the original (ATL_5895) nucleotide. Hard randomization was performed using degenerate oligonucleotides containing NNS codons. Three rounds of phage display were performed for each library. Phage display was performed against immobilized human HTT exon 1 48Q GST protein or biotinylated PQPQPPPPPPPPPP peptide in solution. The phage library was incubated with antigen and then washed to remove unbound phage. Bound phage were then eluted using trypsin (selection) or IgG elution buffer (biotinylated peptide selection). TG1 cells were infected with eluted phage and then plated on selective medium. Colonies from the second and third rounds were sequenced, and phage ELISA against at least one antigen was performed to assess binding of the phage clones. Sequences with improved binding in phage ELISA compared to ATL_5895 were expressed in IgG1 format.

[0221] indirect ELISA Indirect ELISA was performed using HTT exon-1 48Q GST. The irrelevant protein lysozyme was used as a control. Each construct was coated onto a Nunc Maxisorp plate at 5 μg / ml in PBS and incubated overnight at 4°C. The plate was blocked with PBS + 3% nonfat dry milk at room temperature for 1 hour. The buffer was discarded, and the plate was washed three times with PBS + 0.1% Tween-20. Eight-point, three-fold serial dilutions (starting at 60 μg / ml) of test or control antibodies were added to the wells in PBS + 3% nonfat dry milk and incubated at room temperature for 1 hour. For each antigen, wells received 50 μl buffer only (blank control). The plate was incubated at room temperature for 1 hour. The buffer was discarded, and the plate was washed three times with PBS + 0.1% Tween-20. HRP-conjugated secondary antibody in PBS + 2% nonfat dry milk was added to the plate 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. TMB substrate was added to each well and developed for 2 minutes at room temperature. The reaction was stopped with 0.5% sulfuric acid. The absorbance at 450 nm was read for each well.

[0222] Seeding assay To determine the ability of antibodies to bind seed-competent HTT species and affect HTT aggregation, the aggregation rate of FRET-tagged recombinant HTT was assessed by the 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 C-terminally fused to CyPet or YPet (GST-Ex1Q48-CyPet or GST-Ex1Q48-YPet) were produced in Escherichia coli (E. coli) BL21-CodonPlus-RP and affinity-purified on glutathione-Sepharose beads. The purified proteins were dialyzed overnight at 4°C against 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 5% glycerol, flash-frozen in liquid N2, and stored at -80°C.

[0223] Frozen R6 / 2 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 complete protease inhibitor cocktail. The homogenate was incubated on a rotating wheel at 4°C for 1 h and centrifuged at 2,700 × g (4°C) for 20 min to remove cellular debris.

[0224] 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 results in a time- and concentration-dependent increase in FRET. Aggregation was tested in the presence of 10 nM fibrils generated from recombinant HTT and 2.5 μg of brain homogenate obtained from R6 / 2 mice. Immunodepletion of seeds with the disclosed antibodies ATL_0005895 and ATL_0005901, as well as MW8 (Millipore, MABN2529) and MW1 (Millipore, MABN2427), was tested. Immunodepletion was performed using 25 μl of protein G beads (Life Technologies) and 6 μg of antibody.

[0225] In vitro selectivity assay To assess antibody selectivity, ATL_5895, ATL_5901, and ATL_5567 were screened for binding against fixed HEK293 cells expressing 6105 individual full-length human plasma membrane proteins, secreted human secreted proteins, and cell surface-tethered human secreted proteins, as well as an additional 400 human heterodimers, followed by a series of confirmatory screens, all performed on the Retrogenix cell microarray platform (Charles River). Because HTT was not a protein in this screening panel, it was spotted onto screening slides as an antigen in gelatin as a positive control for the fixed version of the assay.

[0226] Live animal PET / CT scanning to assess pharmacokinetics and brain penetration To assess the pharmacokinetics and brain penetration of ATL_5895, a PET-labeled version of the antibody was prepared and PET was performed in live animals as well as gamma counting of postmortem tissues.

[0227] ATL-5895 was radiolabeled with zirconium-89 (89Zr) in a two-step procedure: 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). 2. DfO-ATL-5895 was then radiolabeled with Zr at room temperature, and the final product Zr-DfO-ATL-5895 was purified by SEC, after which a 20 μL aliquot of Zr-DfO-ATL-5895 was injected into a size-exclusion HPLC system to allow assessment of radiochemical purity and DfO-ATL5895 concentration.

[0228] Transgenic female R6 / 1 mice (Jackson Laboratory stock number 006471) aged 11–12 weeks and 14–15 weeks were used, along with age-matched C57BL / 6J controls. Mice were administered 100 μl of 89Zr-Df-ATL5895 at 1.5 ± 0.3 MBq (1.15 mg / kg). PET / CT scans were performed under anesthesia (1.5–2.5% isoflurane), and static PET images were acquired using a Molecubes β-CUBE at 1, 24, 48, 72, and 168 hours post-injection. CT scans were performed after each PET scan using a Molecubes X-Cube. Image analysis was performed using PMOD software.

[0229] Blood samples (20 μl) were collected by the capillary tail method at 0, 1, 6, 12, 24, 48, 72, and 168 hours post-dose and assessed for 89Zr-Df-ATL5895 levels by gamma counting. Ex vivo tissue gamma counting was performed at 168 hours post-dose. A Perkin Elmer Wallac Wizard gamma counter was used for ex vivo organ biodistribution data.

[0230] Manufacturability - ATL5895 Thermostability study. ATLX_1095 (ATL_5895) expressed from CHO cells and purified in one step at 5 mg / mL in 20 mM histidine-acetate, 150 mM NaCl, pH 5.5 was subjected to temperatures of -80°C, +4°C, +21°C, and +40°C for 4 weeks.

[0231] Ten 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 over 10 cycles within 24 hours.

[0232] Protein Thermal Shift and Light Scattering. Protein thermal shift measurements were performed in triplicate on an Uncle (Unchained labs) for unstressed ATLX_1095 (ATL_5895) at 5 mg / mL in 20 mM histidine-acetate, 150 mM NaCl, pH 5.5. 8.8 μL of sample was loaded into three wells of a Uni (Unchained labs - a suitable strip of 16 9 μL quartz cuvettes placed inside a blue metal frame with a silicone seal). Laser settings were set to achieve an initial fluorescence in the 300-350 nm range of 10,000-50,000 counts. The antibody was ramped from 25°C to 95°C at a rate of 0.5°C / min, with excitation at 266 nm while simultaneously monitoring fluorescence emission and SLS. Melting temperatures (Tm1 / Tm2) and aggregation temperatures (Tagg / Tonset) were analyzed using Uncle Analysis software v6 (Unchained Labs). Tm measurements were calculated from the centroid mean (BCM) of the fluorescence intensity curves from 300 to 430 nm, while Tagg and Tonset were calculated from the intensity of light scattered at 266 nm.

[0233] 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. For reduced samples, 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. Injection was performed at 4600 V for 20 seconds, and for reduced samples, separation was performed at 5750 V for 25 minutes. Results were analyzed using Compass for iCE software (Bio-Techne) and Chromeleon software (Thermo).

[0234] cIEF. cIEF analysis was performed using Maurice (ProteinSimple, Bio-Techne). A 5 mg / mL ATLX_1095 (ATL_5895) sample 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 achieve a final antibody concentration of 0.15–0.25 mg / mL. Sample separation was performed at 1500 V for 1 min, followed by 3000 V for x min. Results were analyzed using Compass for iCE software (Bio-Techne) and Chromeleon software (Thermo).

[0235] 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 μg of sample was loaded onto either a Zorbax GF-250 SEC-HPLC column (Agilent) or a TSKgel G3000SWxl column (TOSOH Bioscience) on a Vanquish Flex (Thermo) by injecting 25 μL of 1 mg / ml sample. Samples were eluted isocratically with 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 (Zorbax column) or 40 minutes (TSKgel column). Chromatograms were integrated and monomer purity was determined using Chromeleon software (Thermo).

[0236] HTT Sandwich ELISA. To evaluate the binding potency of stressed antibody samples against HTT protein, antibodies were evaluated in a sandwich ELISA format for binding to HTT exon 1 containing the 48Q repeat (see Table 1 for antigen sequence). 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 per well of a 96-well plate and left overnight at 4°C. 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 for 1 hour at room temperature on a plate shaker (300-400 rpm). The plate was washed with PBS / 0.1% Tween. Eight-point, 3-fold serial dilutions (starting at 60 μg / mL) of test and isotype control antibodies were added to the plate. The plate was incubated for 1 hour at room temperature on a plate shaker (300-400 rpm). The plate was washed with PBS / 0.1% Tween. 10 anti-human IgG HRP (80 ng / mL, Jackson ImmunoResearch, #109-035-097) was added per well. The plate was washed with PBS / 0.1% Tween, 50 μL of TMB solution (Lifetechnology, #002023) was added, and the plate was incubated for 6-9 minutes at room temperature in the dark. 50 μL of stop solution (0.5 M sulfuric acid) was added to the plate (Fisher Chemical, #12933634). Absorbance was read on a CLARIO Star at 450 nm. Data analysis was performed using GraphPad Prism 10 software (10.1.0.316). EC50 (half-maximal concentration) values ​​were calculated using unconstrained, nonlinear, four-parameter curve fitting.

[0237] Preparation of ATL_5895 for solubility evaluation. Recombinant antibody ATL_5895 was transiently expressed in ExpiCHO-S cells using Expifectamine reagent (Thermo) according to the manufacturer's protocol. Transfectants were cultured with feed at 32°C for 13 days, after which the cells were removed and the supernatant was harvested by mixing with diatomaceous earth (Sartorius) and filtering through a 0.22 μm PES membrane. The collected supernatant was purified using Protein A chromatography eluting with 50 mM sodium acetate, pH 3.6. Elution fractions were pooled and buffer-exchanged into 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5, and stored at 4°C, followed by long-term storage at -80°C.

[0238] 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 μL samples were collected from the sample reservoir at three time points. Sample concentrations were determined by absorbance at 280 nm by UV / Vis spectrophotometry in Lunatic (Unchained Labs). Samples were evaluated for aggregation by SEC-HPLC. Antibody samples were diluted to approximately 1 mg / mL with 20 mM histidine acetate, 150 mM NaCl, pH 5.5, and filtered through a 0.22 μm filter. Approximately 25 μg of sample was loaded onto a TSKgel G3000SWxl column (TOSOH Bioscience) on a Vanquish Flex (Thermo) by injecting 25 μL of 1 mg / mL sample. Samples were isocratically eluted with 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 (Zorbax column) or 40 minutes (TSKgel column). Chromatograms were integrated and monomer purity determined using Chromeleon software (Thermo).

[0239] Mouse HTT ELISA To evaluate the binding potency of antibodies against mouse HTT protein, ATL5895, ATL6376, and ATL6377 were evaluated in a direct ELISA format for binding to mouse HTT and human lysozyme (as a negative control antigen). Mouse HTT antigen (SEQ ID NO: 177) or lysozyme was directly absorbed onto ELISA plates at 3 μg / ml (50 μl / well) and incubated overnight at 4°C. The plates were washed with PBS. The plates were blocked with 200 μl / well of blocking solution (1% BSA w / v in PBS) for 1 hour at room temperature. Following this, the blocking solution was removed, and the antibody to be evaluated was diluted in a dilution series (1 μM to 0.05 nM) in blocking solution (1% BSA w / v in PBS) and applied to the plate. The plate was incubated for 1 hour at room temperature. The plate was then washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch, #109-035-097) was added to the plate and incubated for 1 hour at room temperature 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 for 5 minutes at room temperature before adding stop solution (0.5 M sulfuric acid). Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader. Analysis was performed in GraphPad Prism using a nonlinear fit curve fitting algorithm, and EC50 values ​​were calculated.

[0240] Example 1 – Convergence analysis of AD cohorts to identify VH sequences associated with resilience Convergent sequence clusters derived from the antibody repertoires of resilient populations can be used to identify disease-specific antibody sequences. In the case of neurodegeneration, resilience can be defined as the prolonged absence of symptoms despite a strong disease predisposition.

[0241] Using cognitive scores and biomarkers (in collaboration with the European Prevention of Alzheimer's Dementia (EPAD)), we identified candidate protective antibodies from a resilient subgroup of at-risk patients from a cohort of patients at risk for dementia. Figure 1A shows the workflow used in this example to identify convergent VH sequences from an AD dataset. Resilience was defined as patients at risk for Alzheimer's disease (AD) who had significantly reduced β-amyloid in their cerebrospinal fluid (CSF) compared to healthy controls, which strongly correlates with increased β-amyloid deposition in the brain. This subgroup of patients also had lower pTau levels in their CSF, indicating significantly less neuronal damage compared to AD progressors, and demonstrated continued normal cognitive function compared to age-matched progressors with adverse β-amyloid and p-Tau CSF biomarkers.

[0242] We identified one cluster of related antibody heavy chains that converged across resilient individuals but were absent from control individuals (Figure 1B). In Figure 1B, the sequence labeled "known HTT binder" is the sequence labeled ATL_0005059 herein, also referred to as NI-302.8F1, and described in U.S. Patent No. 11,401,325 B2. It is important to note that the antibodies described herein were identified through a process independent of this previous antibody. Indeed, the present antibodies were identified as related antibody heavy chains that converged across individuals resilient to neurodegenerative disease. These antibodies were identified to bind HTT and were therefore aligned to known antibodies for this context. In other words, the presently described antibodies were not developed by adapting existing antibodies. Rather, the presently described antibodies were discovered through the analysis and optimization of naturally occurring protective (and therefore likely therapeutically effective) antibodies. Their identification was entirely target-specific and sequence-independent, and their optimization was completely independent of prior art antibodies; instead, they further improved upon already superior antibodies derived from naturally occurring sequences. These antibodies exhibited immune activation signatures, supporting their role in resilience. To deconvolute the targets of this cluster of VHs, they were compared to a database of antibodies with known binding specificities curated from the literature. One VH within the cluster shared a CDR3 amino acid sequence identical to a known binder of huntingtin, a protein encoded by the Htt gene strongly associated with Huntington's disease. This finding indicated that convergent VHs in the cluster may also bind to the HTT protein.

[0243] Two of these VHs (ATL5060 and ATL5061 - see sequences in Table 1 and Figure 1B) were expressed as antibodies using light chains (VLs) from known binders, and both antibodies were confirmed to bind to HTT by ELISA.

[0244] The discovery of antibodies against HTT in patients "at risk" for resilient AD may be considered unexpected. Aggregated and mutant HTT (mHTT) protein has been reported as a primary cause of Huntington's disease, but the presence of mutant or aggregated protein has been associated with other neurodegenerative disorders. HTT aggregates accumulate in the cytoplasm of dystrophic neurons and microglia in AD brains (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 neurons of the hippocampus and prefrontal cortex in another AD study (Axenhus, M, et al. Huntingtin Levels Are Elevated in Hippocampal Post-Mortem Samples of Alzheimer's Disease Brain. Curr Alzheimer Res 17, 858 (2020)). 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)). Furthermore, CAG codon expansions (CAG>40) in Htt have been reported in a small proportion of patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), a percentage 4.4 times higher than in healthy individuals (Dewan, R. et al. Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis. Neuron 109, 448 (2021)).Furthermore, mHTT polyglutamine expression has been shown to increase the seeding properties of aggregated TDP-43 in cellular 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)). Thus, HTT dysfunction may play a pathogenic role in a broader set of neurodegenerative diseases than previously thought and at an earlier stage of pathology than alterations mediated by tau, beta-amyloid, or TDP-43.

[0245] In addition to the two individuals from the EPAD cohort, homologs were found in another individual with AD from a separate cohort and in two individuals who were genetically predisposed to FTD but expressed resilience to it (data not shown), further supporting a role for mHTT in various neurodegenerative conditions.

[0246] Example 2 - Phage display to obtain optimal VJ / VL pairing Based on the discovery of antibody ATL_5060, combinatorial phage display was used to identify optimal VL partners for VH. The VH sequence from ATL_5060 was combined with over one million potential VL partners in a phage display library, followed by selection against HTT protein to enrich for functional binders. This process is shown in Figures 2A and 2B.

[0247] Phage selection yielded seven unique scFv sequences (designated ATL_0005331-5337), and mHTT phage ELISA suggested 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). Both assays shown in Figures 3A and 3B are indirect ELISAs for measuring binding to mHTT coated on a plate. However, the test samples differ between A and B. In A, the test sample is a phage displaying an antibody fragment corresponding to the selected antibody sequence. In B, the test sample is an antibody sequence formatted as human IgG. The experiment shown in A is a screening experiment in which a higher concentration of mHTT is used to ensure detection of binding levels. The experiment shown in B is a determination of the binding equilibrium, where the concentration of mHTT used provides increased sensitivity. The minimal difference in absorbance observed in A may be due to the variability of phage in each test sample, including different display levels of antibody fragments and different concentrations of phage in the test samples applied to the experiment, resulting in varying concentrations of antibody fragments in the test samples. In contrast, in B, there is little variation in the amount of antibody used in the test samples, as this can be easily determined based on protein concentration.

[0248] ATL_5331, ATL_5334 and ATL_5335 were selected for further optimization.

[0249] Figure 4A shows the convergent VH sequence identified in an AD-resilient individual (ATL_0005042). Figure 4B shows the functionally paired VL (ATL_0005331-5335) aligned to CA_0000274 VL (also referred to herein as ATL_0005059 or NI-302.8F1 and described in U.S. Pat. No. 11,401,325 B2) from phage display selection and sequence analysis in HTT exon 1.

[0250] Example 3 – Epitope Mapping To evaluate the epitopes of the antibodies against the HTT protein (ATL_5331, ATL_5566 (=ATL_5334 with the free cysteine ​​removed) and ATL_5335), the antibodies were evaluated against peptide arrays constructed from peptide fragments corresponding to sequences of human, cynomolgus monkey (cyno, two different reference sequences for cynomolgus monkey HTT that are likely to be the HTT protein sequence) and mouse HTT exon 1.

[0251] The peptide array consisted of 15-mer linear peptides with a 14 amino acid overlap across HTT exon 1 (see Figure 5), allowing for high-resolution epitope mapping. Species cross-reactivity was tested in parallel using two cynomolgus monkey and one mouse isoform of HTT exon 1.

[0252] Table 2 shows the sequences used to generate the peptides in the array.

[0253] [Table 14]

[0254] The resulting huntingtin peptide microarrays contained duplicated HTT peptides and were framed by additional HA (YPYDVPDYAG, 48 spots) and polio (KEVPALTAVETGAT, 48 spots) control peptides. Huntingtin peptide microarrays were incubated with antibody samples at concentrations of 1 μg / ml and 10 μg / ml, followed by staining with secondary antibodies (goat anti-human IgG (H+L) DyLight680 at 0.2 μg / ml) and control antibodies (mouse monoclonal anti-HA (12CA5) DyLight800 at 0.2 μg / ml) and scanning with an Innopsys InnoScan 710-IR microarray scanner. Spot intensity quantification and peptide annotation were performed using the PepSlide® Analyzer. Briefly, spot intensity quantification and peptide annotation were based on 16-bit grayscale tiff files. Microarray image analysis was performed using a PepSlide® Analyzer. The fluorescence intensity of each spot was decomposed into raw, foreground, and background signals, and the median foreground intensity (referred to as the "corrected intensity") and spot-to-spot variation of spot replicates were calculated. A maximum spot-to-spot variation of 40% was allowed; otherwise, the corresponding intensity value was set to zero. Overall spot intensity was visualized using a plot of the average spot intensity of an assay using a human antibody sample against the antigen sequence from the N-terminus of human huntingtin protein to the C-terminus of mouse huntingtin protein. The intensity plot was correlated with peptide and intensity maps and visual inspection of the microarray scan to identify the epitope of the antibody sample.

[0255] Figures 6-8 show the binding strength of three selected monoclonal antibodies (mAbs) to regions of HTT exon 1. All mAbs showed similar binding profiles across different species, with binding primarily observed at two distinct sites in HTT exon 1. Alignment revealed consensus motifs for the major antibody responses (shown in bold in Figures 6-8 (lower panels)). The consensus motif for binding shared between binding sites on exon 1 was identified as [P / Q]Q[P / Q]QPPPPPPPPPPP (SEQ ID NO: 113).

[0256] Table 3 is a summary table of peptides with binding detected for at least one of the antibody concentrations.

[0257] [Table 15]

[0258] [Table 16]

[0259] [Table 17]

[0260] In conclusion, epitope mapping suggests that the three selected antibodies have a composite binding site in HTT exon 1 and preferentially bind to peptides with multiple C-terminal prolines.

[0261] Example 4 – Stability Studies To evaluate the stability profile of HTT exon-1 mAbs, a 3-week accelerated stability study was performed on the three selected mAbs mentioned above and their full germline counterparts: ATL_5331, ATL_5334, ATL_5335, ATL_5555 (ATL_5331 full germline), ATL_5556 (ATL_5334 full germline), and ATL_5557 (fully germlined ATL_5335). The workflow of the stability study is shown in Figure 9.

[0262] Antibodies were stored at -80°C, 4°C, room temperature, or 40°C for 3 weeks, and samples were subjected to five repeated freeze-thaw cycles from -80°C to room temperature. Quality control analyses followed, including assessment of purity, aggregation, degradation, charge variants, and thermal stability.

[0263] Protein aggregation during antibody storage must be kept to a minimum as it can cause immunogenic reactions. Size exclusion chromatography (SEC-HPLC) was used to assess antibody purity and aggregation in a 3-week stability study and after five freeze-thaw cycles.

[0264] Figure 10 shows that no soluble aggregates form after 3 weeks of incubation at -80°C, 4°C, room temperature (21°C), and 40°C (SEC-HPLC). Table 4 further supports the excellent stability of the six tested antibodies, showing low levels of high molecular weight species (HMWS) and low levels of low molecular weight species (LMWS), indicating low aggregation and degradation tendency of the antibodies up to 40°C, which is further supported by the high percentage of monomer in each of over 95% of the samples throughout the study.

[0265] [Table 18]

[0266] No detectable soluble aggregates were formed for antibodies ATL_5331 or ATL_5335 after a low pH hold step (pH 3.5) of up to 120 minutes (SEC-HPLC) (Figure 11 and Table 5).

[0267] [Table 19]

[0268] 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 ​​were within the typical range of 7.5 to 9 for antibodies suitable for downstream processing.

[0269] [Table 20]

[0270] An increase in acidic charge variants was observed at the highest temperature stress condition after 3 weeks of incubation at 40°C, but not at any of the other temperatures tested (Figure 12B) or after five freeze-thaw cycles (Figure 12C). In conclusion, the antibody does not undergo significant changes in biophysical properties compared to accelerated temperature stress studies performed in non-optimized formulation buffer at 5 mg / ml.

[0271] Example 5 – In vivo PK profile To determine the serum PK of the lead antibody, in vivo PK experiments were performed using ATL_0005335 (the parent antibody of ATL_0005567). Antibody levels were assessed by ELISA in serum samples collected 1, 4, 8, 24, 72, and 144 hours after administration. ATL_0005335 exhibited linear PK and no altered clearance compared to a non-binding IgG1 antibody in wild-type animals. This suggests that the antibody does not unexpectedly bind to molecules outside the CNS that would affect pharmacokinetics and potentially safety.

[0272] Example 6 – Lead antibody optimization To identify optimal candidates for preclinical and ultimately clinic testing, antibodies were subjected to various rounds of triage, as shown in Figure 14. To identify lead antibodies with optimal developmental characteristics, an initial antibody panel was designed, consisting of variants of ATL_5331, 5334, and 5335, including germline intermediate mutations and liability-eliminating mutations. While reversion to germline framework sequences reduces the potential immunogenicity of the antibody, mutating sequence motifs to eliminate potential liability may help minimize future manufacturing and pharmacokinetic issues caused by amino acid modifications.

[0273] Tables 7 and 8 show the VH and VL sequences for the parent antibodies, their germline equivalents and equivalents with additional occurring mutations.

[0274] [Table 21]

[0275] [Table 22]

[0276] [Table 23]

[0277] [Table 24]

[0278] [Table 25]

[0279] A total of 69 mAbs (monoclonal antibodies) were triaged primarily based on binding to HTT exon 1 in ELISA, and thermal stability analysis was determined by measuring Tm using SYPRO™ Orange in a thermal shift assay (see Figure 15). There was no difference in thermal stability with similar Tm for all variants (Tm1 (mean) = 62.4 ± 2.1 °C (% RSD)) (Figure 15). Comparing the binding ELISA, all 5331 variants were ranked highly, whereas 5334 variants had variable rankings and 5335 variants were ranked low.

[0280] Subsequently, a second mutant panel containing the selected mutation combinations was designed, after which antibodies were further triaged based on their binding to HTT exon 1 and thermostability to select lead antibodies.

[0281] Lead antibodies were selected primarily based on affinity for HTT (using a single-point binding ELISA) and binding potency as determined by sandwich HTT ELISA (ranking 48Q HTT Exo-1 EC50, E50<50nM, then EC50(25Q:48Q) and 48Q), and then based on germline mutations and liability-eliminating mutations (low-yielding mutations, removal of stability flags, etc., e.g., G54A eliminates the aspartate isomerization motif).

[0282] Antibodies with lower EC50 values ​​have greater binding potency, and therefore affinity, for HTT exon 1, as determined by HTT sandwich ELISA. Initially, antibodies were ranked based on their EC50 values ​​for binding to 48Q HTT exon-1. Antibodies with an EC50 of less than 50 nm were triaged and further ranked based on EC50 and 25Q:48Q ratio. The higher the ratio of 25Q:48Q binding, the more potent the antibody is for 48Q HTT relative to 25Q HTT (i.e., the lower the 48Q ec50, the better the 25Q ec50). Additional factors considered for lead antibody selection were the number of germline mutations and liability-eliminating mutations, as these antibodies pose fewer safety risks.

[0283] Finally, three lead antibodies were selected: ATL_5901 (5334 variant, rank 1), ATL_5895 (5331 variant, rank 1), and ATL_5567 (5335 variant, rank 3).

[0284] ATL_5901 and ATL_5895 had similarly low EC50 levels (see Figure 16 and Tables 9-10) and included developmental and germline mutations. ATL_5567 was the best-performing 5335-derived mutant.

[0285] [Table 26]

[0286] [Table 27]

[0287] [Table 28]

[0288] All three lead antibodies consistently showed greater binding potency for HTT exon 1 25Q (up to 9-fold) (Table 9), HTT exon 1 48Q (Table 10), and showed higher 25Q:48Q ratios (Table 11) when compared to the reference antibody NI-302.8F1 described in U.S. Pat. No. 11,401,325 B2.

[0289] Thus, these data indicate that the three lead antibodies, ATL_5901, ATL_5895, and ATL_5667, have improved binding properties compared to prior art antibodies. The lead antibodies have also been optimized with respect to germline and sequence liability-removing mutations to potentially reduce immunogenicity and increase shelf life, stability, and suitability for manufacturing.

[0290] The suitability of ATL_5895 for manufacturing was further verified by verifying that no aggregation was observed after 4 weeks of incubation at 40°C (vs. -80°C) (by SEC-HPLC), as described above, and that no aggregation was observed after 10 freeze-thaw cycles (by SEC-HPLC), as described above. ATL_5895 also has a pI within the appropriate range for downstream processing (i.e., 7.5E-9).

[0291] Example 7 – Binding to disease-associated HTT Huntington's disease is caused by a pathological expansion of a cytosine-adenine-guanine (CAG) triplet repeat in the huntingtin (HTT) gene, resulting in the production of mutant HTT protein. As shown in Example 6, ATL_5331, 5334, 5335, 5895, 5901, and 5567 all bind to 48Q HTT exon 1 (i.e., mutant HTT) by ELISA, indicating that the antibodies can bind to pathologically long forms of HTT. To determine whether the antibodies can bind to high-molecular-weight HTT (suggesting a more pathological, aggregate-like state of HTT), immunoprecipitation experiments were performed using ATL_0005335 (the parent antibody of ATL_0005567).

[0292] To confirm that the antibodies of the present invention can bind to disease-associated HTT, we isolated a high molecular weight species of HTT (HTT) that expresses 110 CAG repeats and resembles the mutant HTT protein. 110 The binding of ATL_5335 to the U-2 OS cell line, which carries HTT (HTT), was examined by Western blot after immunoprecipitation with ATL_5335. As shown in Figure 17A, ATL_5335 binds to HTT (HTT), which has a molecular weight of more than 180 kDa. 110 ) can be immunoprecipitated.

[0293] R6 / 2 transgenic mice express the 5' end of the human HTT gene, including exon 1 with approximately 120 CAG repeats, and exhibit a neurological phenotype similar to that of HD in humans, including the production of pathological HTT aggregates. Figure 17B shows that ATL_0005895, ATL_0005901, and ATL_0005567 can immunoprecipitate high molecular weight HTT (in this case, >250 kDa, the highest molecular weight protein marker used in this gel ladder) from brain homogenates from R6 / 2 mice. Therefore, these data demonstrate that the antibodies of the present invention can bind to aggregated HTT protein.

[0294] To confirm that ATL_0005895 can bind to disease-associated HTT in the 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 blot. High-molecular-weight HTT material was successfully immunoprecipitated by ATL_0005895, but not by an isotype control antibody (Figures 17C and 17D). Notably, Figure 17C shows that ATL_0005895 can immunoprecipitate high-molecular-weight HTT from human brain homogenates derived from Huntington's disease patients, as quantified in Figure 17D. These data demonstrate that the antibodies of the present invention can bind to highly disease-associated aggregated HTT protein from human brain tissue.

[0295] Example 8 - Binding of anti-HTT antibodies to seed-competent HTT seeds Self-propagating protein aggregates drive pathogenesis in many neurodegenerative diseases, including HD, which is characterized by the pathological aggregation of toxic mHTT species. Therefore, it was important to test the ability of ATL_5895 and ATL_5901 to bind to these seed-competent HTT species and inhibit pathological HTT aggregation.

[0296] To test the anti-seeding ability of the anti-HTT antibodies ATL_5895 and ATL_5901, the aggregation kinetics of FRET-tagged recombinant HTT was assessed by a FRET-based mHTT aggregate seeding (FRASE) assay as described by 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.

[0297] Figure 18 shows the results of a FRASE assay using recombinant HTT seeds (Figure 18A) and brain homogenates from R6 / 2 mice (Figure 18B). Immunodepletion of pathological HTT or "seeds" with antibodies ATL_0005895 and ATL_0005901 reduced the in vitro aggregation rate of seeding-competent HTT seeds. Notably, MW8 (Millipore, MABN2529), a murine monoclonal IgG2A antibody against human HTT, binds to aggregated HTT and achieves this effect to some extent, whereas MW1 (Millipore, MABN2427), which binds to the polyQ region of HTT, does not (see Figures 18A and B).

[0298] Taken together, these results demonstrate that the antibodies described herein can reduce the rate of self-propagation of toxic HTT species, suggesting that these antibodies may slow disease progression by reducing HTT seeding.

[0299] Example 9 – Phagocytosis Assay As explained above in Examples 7 and 8, HD is characterized by the aggregation of mHTT species, which have the ability to self-propagate / seed, thereby causing the pathogenesis of HD. An important immunological defense mechanism in the central nervous system is the phagocytic clearance of neurotoxic proteins, such as these mHTT species, by microglia.

[0300] To investigate whether binding of the anti-HTT antibody ATL_5895 to exon 1 of HTT improves phagocytic clearance of HTT by microglia, we monitored phagocytic activity in cultures using an in vitro assay using induced pluripotent stem cell (iPSC)-derived microglia (see the Materials and Methods section above).

[0301] Figure 19 shows the results of an assay using the anti-HTT antibody ATL_5895 versus the isotype control antibody ATL5338, which binds fluorescein. Figure 19 shows that binding of ATL_5895 dose-dependently increases the uptake of 48Q HTT-coated beads in iPSC microglia compared to beads treated with the isotype control.

[0302] The results of this experiment demonstrate that the antibodies described herein increase the phagocytic clearance of toxic mHTT species.

[0303] Example 10 – In vivo PK studies The pharmacokinetic properties of a selection of anti-HTT antibodies were tested in vivo to assess circulating levels of the antibodies as well as their ability to penetrate the CNS.

[0304] Antibody levels were assessed by ELISA in serum samples collected 1, 4, 8, 24, 72 and 144 hours post-dose and in CSF samples collected 4 and 144 hours post-dose.

[0305] Figure 20 shows the results of a PK study. ATL_5901 and ATL_5567 exhibit linear serum PK profiles when administered at a concentration of 10 mg / kg (Figure 20A). Figure 20B shows the serum concentrations of ATL_5895 after treatment with 1, 10, or 60 mg / kg of ATL_5895, all of which result in linear serum PK profiles. Importantly, ATL_5895 also exhibits evidence of CNS penetration (Figure 20C). Typical penetration of IgG1 in the CNS is 0.1-0.3%. This data demonstrates that CNS exposure above the EC50 of the antibody is achievable.

[0306] A proof-of-concept pharmacology study in the R6 / 1 mouse model is described in Example 15.

[0307] Example 11 – Affinity maturation ATL_5895 As demonstrated in the above examples, ATL_5895 binds to disease-associated HTT, inhibits seeding, and increases phagocytic clearance of pathological HTT. Therefore, we investigated whether there was scope for affinity maturation of the ATL_5895 antibody to identify additional high-affinity HTT-binding antibodies.

[0308] Affinity maturation of ATL_5895 was performed by phage display using a phage library containing one or more mutants of the following amino acids: the YCIPPPYYYYYGLDV sequence in the VH of ATL_5895 ("extended CDR3H") and the GSYAGTANV sequence in the VL of ATL_5895 (CDR3L). "Extended CDR" refers to the CDR3H as well as positions outside the CDR3H, in this example, a region encompassing four amino acids. In this case, 103 was one of the positions selected for "hard randomization" (described above under "Phage Display" in the "Materials and Methods" section). To introduce more subtle changes in antibody function, residues close to but outside the CDRs were mutated. Three rounds of phage selection were performed against human HTT exon 1 48Q or biotinylated GYSLPQPQPPPPPPPPPP peptide. The extended CDR3H and CDR3L regions of the antibodies identified through affinity maturation by phage display are shown in Table 12. The complete VH and VL sequences of these antibodies are shown in Table 1.

[0309] [Table 29]

[0310] Figures 21 and 22 show the results of indirect and sandwich ELISA, respectively, for the binding of the newly identified antibodies to exon 1 48Q HTT. Table 13 shows the EC50 values ​​obtained from the sandwich ELISA results in Figure 22.

[0311] [Table 30]

[0312] The above data demonstrate that additional antibodies identified through phage display can bind exon 1 48Q HTT with high affinity.

[0313] Antibodies ATL_6205, ATL_6202, ATL_6194 and 6203 were used as the basis for further optimization in Example 17. Antibody ATL_6195 was also selected for further investigation because a homologous sequence was found in resilient patients with HD.

[0314] Example 12 – HD patients produce HTT-binding antibodies To identify additional anti-HTT-binding antibodies, the inventors studied 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 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.

[0315] Two antibodies with homology to ATL_5895 were identified within the same ENBB patient, and they were 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 also generated that contained only the HCDR3s of the two homologous antibodies, with the remainder of the antibody being ATL5895. Table 14 contains details regarding the VH and VL pairings and CDR3 usage of the newly generated antibodies.

[0316] Table 16 shows the VH CDR sequences for each of these antibody variants, along with the VH CDR sequences of ATL5895 and ATL5901. The complete VH and VL sequences of these antibodies are shown in Table 1.

[0317] [Table 31]

[0318] Figure 23 shows the results of sandwich ELISA for the binding of each of the newly identified antibodies to exon 1 48Q HTT. All antibodies were shown to bind to HTT, except for the control antibody 5338 (isotype control, conjugated to fluorescein). Table 15 shows the EC50 values ​​obtained from the sandwich ELISA results shown in Figure 23.

[0319] [Table 32]

[0320] Together, these data confirm that the antibodies of the invention are capable of binding disease-associated HTT.

[0321] [Table 33]

[0322] Figure 24 shows the sequences of the FW regions and CDRs for the VH (Figure 24A) and VL (Figure 24B) of several antibodies of the disclosure.

[0323] Example 13 – In vitro selectivity assay To examine antibody selectivity, ATL_5895 and ATL_5567 were first subjected to a library screen for binding to fixed HEK293 cells overexpressing 6105 individual full-length human plasma membrane proteins, secreted human secreted proteins, and cell surface-tethered human secreted proteins, as well as to an additional 400 human heterodimers to identify library interactions. Following this library screen, a series of confirmation screens were performed in which all library interactions were re-expressed in fixed and live cells and probed with each test antibody or control treatment to determine which interactions were reproducible and specific to each test antibody (see the "Materials and Methods" section above). This was performed on both fixed and live cells. HTT was spotted on screening slides as an antigen in gelatin as a positive control (since HTT was not part of this particular screening panel).

[0324] Both ATL_5895 and ATL_5567 bound strongly to the HTT positive control (2 repeats per condition, 2 μg / mL antibody for ATL5895, 5 μg / mL antibody for ATL5567 and the isotype control ATL5338). Another polyQ protein, CACNA1A, was present and not detected by either antibody. ATLX-1095 and ATL5567 showed no confirmed hits in this screening panel, demonstrating their selectivity. Rituximab was used as a positive control for CD20 and hit this antigen as expected.

[0325] This data demonstrates that the ATL_5895 and ATL_5567 antibodies described herein specifically bind to HTT.

[0326] Example 14 – Live animal PET / CT scans to assess pharmacokinetics and brain penetration To assess the pharmacokinetics and brain penetration of ATL_5895, a PET-labeled version of the antibody was prepared and PET was performed on live animals as well as gamma counting of postmortem tissues (see the Materials and Methods section above).

[0327] Figure 25 shows the results of live animal PET experiments and gamma counting assays. Half-lives ranged from 8 to 9 days for the labeled antibody in both C57BL / 6J and R6 / 1 mice in the 11-12 week and 14-15 week cohorts (Figures 25A and 25B). The mean brain:blood ratios, calculated by postmortem gamma counting, were estimated to be 0.03 ± 0.004 at 11-12 weeks for both groups and 0.04 ± 0.01 at 14-15 weeks for both groups (Figures 25C and 25D). As demonstrated in Example 10, standard / classical PK experiments showed an exposure of approximately 5.5 nM in the CSF (Figure 20C), indicating that brain levels in this experiment may be underestimated. Live animal PET showed typical human IgG1 biodistribution of the labeled antibody, with the majority detected in the blood (Figures 25E and 25F) - although, as explained above, the antibody was also detected in the brain and CSF.

[0328] Example 15 – In vivo PD assay R6 / 1 is a transgenic mouse model of Huntington's disease (Hansson et al., Cell, 1996) that displays a progressive neuronal phenotype mimicking many of the features of Huntington's disease, including the accumulation of aggregates over time (Hansson et al., EJN, 2001). These mice ubiquitously express a transgene containing the 5' end of mutant human huntingtin, including approximately 1 kb of 5' UTR sequence, exon 1 (with an expanded CAG repeat of 115–150 CAG repeats), and the first 262 bp of intron 1. To evaluate the effect of ATL_5895 (ATLX_1095) on HTT aggregate burden in R6 / 1 mice, mice were treated with vehicle or ATL_5895 for up to 12 weeks (starting at 5 weeks of age). HTT aggregate burden was assessed in the brains of these mice using an immunoassay (MSD). Soluble mutHTT (2B7, MW1+) was assessed in plasma and an increase was observed, suggesting target engagement and clearance of HTT driven by antibody-HTT complexes in plasma.

[0329] Figure 26 shows the results of a mesoscale discovery (MSD) assay to assess the effect of ATL_5895 (ATLX_1095) on HTT aggregate burden in the striatum and cortex of R6 / 1 mice. As expected, HTT aggregates increased over time in the striatum and cortex of R6 / 1 mice (4C9 / MW8 + HTT, Figure 26A). Concomitantly, there was a decrease in soluble mutant HTT over time, as assessed by the MSD assay (2B7 / MW1). Treatment of R6 / 1 mice with ATL_5895 for 12 weeks resulted in a statistically significant decrease in HTT aggregates (as detected by the MW8 / 4C9 + antibody) in the striatum and cortex (Figure 26B). No reduction in soluble HTT (as detected by the 2B7 / MW1 antibody) or endogenous mouse HTT (as detected by the 2B7 / D7F7 antibody) was observed in the striatum and cortex (Figures 26C and 26D, respectively). Together, these results indicate 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.

[0330] Plasma neurofilament light chain (NEFL) levels were assessed, but no difference was observed between WT and R6 / 1 mice, thus indicating the absence of a rescuing antibody phenotype.

[0331] Example 16 - Manufacturability A stability evaluation of antibody ATLX-1095 (ATL_5895) was also performed, including a 4-week thermal stability study, a 10-cycle freeze-thaw cycle study, thermal stability evaluation (Tm and Tag), and solubility studies. The antibody's biophysical properties were evaluated after exposure to various 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. SEC-HPLC, CE-SDS, cIEF, and ELISA were used to evaluate purity, aggregation, degradation, charge heterogeneity, and changes in binding of stressed antibody samples compared to the -80°C control condition.

[0332] The melting temperature (Tm) and aggregation temperature (Tagg) of the unstressed antibody were assessed by DSF and SLS.

[0333] Antibody solubility was assessed by concentrating ATLX_1095 (ATL_5895) to 89.96 mg / mL using tangential flow filtration (TFF). After concentration and after 1 week at 21° C., samples were assessed for aggregation by SEC-HPLC analysis.

[0334] Protein aggregation during antibody storage must be kept to a minimum because it can cause immunogenic reactions. Size exclusion chromatography (SEC)-HPLC was used to assess antibody purity and aggregation in a 4-week stability study and after 10 freeze-thaw cycles. Figures 27A-B show SEC-HPLC chromatograms of ATL_5895 after 4 weeks of thermal stability and 10 freeze-thaw cycles, respectively. Both show no increase in soluble aggregate formation after incubation at -80°C, 4°C, room temperature (21°C), and 40°C, respectively, for 4 weeks and after 10 freeze-thaw cycles. The results in Table 17 show that monomer purity remained high (>95%).

[0335] [Table 34]

[0336] Capillary isoelectric focusing (cIEF) was used to assess the charge heterogeneity of antibody samples. The isoelectric point (pI) of unstressed ATL_5895 was determined using cIEF. The pI of ATL_5895 was 8.93, within the typical range of 7.5–9 for antibodies 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 assessed by cIEF. An increase in acidic and basic charge variants and a decrease in the main peak were observed at the highest temperature stress condition after 4 weeks of incubation at 40°C, but not at any of the other temperatures tested (Figure 27C) or 10 freeze-thaw cycles (Figure 27D). These results, also summarized in Table 18, show stable % basic and acidic species after most treatments (main isoform % approximately 70%).

[0337] [Table 35]

[0338] Figure 27E and Table 19 show the CE-SDS results for samples of antibody subjected to the indicated treatments (reduced before CE-SDS).

[0339] [Table 36]

[0340] HTT exon-1 sandwich ELISA was performed to assess changes in binding of ATL_5895 to 48Q HTT exon-1 after temperature stress (-80°C, 4°C, room temperature (21°C), 40°C for 4 weeks) and 10 freeze-thaw cycles. Figure 27F shows that there was no significant change in binding to 48Q HTT exon-1 after temperature stress (-80°C, 4°C, room temperature (21°C), 40°C for 4 weeks) and 10 freeze-thaw cycles compared to the isotype control antibody (ATL_5338-011).

[0341] Protein thermal shift and light scattering assays were performed to determine the melting temperature (Tm1 / Tm2) and aggregation temperature (Tagg / Tonset) of ATL_5895 (see the "Materials and Methods" section above). Figure 27G shows the results of the thermal stability assay, summarized in Table 20. These results demonstrate that the ATL_5895 thermal stability parameters are within the typical range for an IgG1 antibody.

[0342] [Table 37]

[0343] A solubility study was performed to assess the solubility of ATL_5895 at different concentrations (see the Materials and Methods section above). After concentration up to 89.96 mg / mL using tangential flow filtration (TFF) and incubation at 21°C for 1 week, no increase in aggregation was observed for ATL_5895 (Figures 27H, 27I). Monomeric purity remained high (>97%) across all samples tested (Tables 21, 22).

[0344] [Table 38]

[0345] [Table 39]

[0346] Example 17 –Affinity matured HTT antibody combinations Antibodies were generated using combinations of mutations present in affinity matured variants ATL_6205, ATL_6202, ATL_6194 and / or ATL_6203 (see Example 11 above).

[0347] 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 the "Materials and Methods" section above). Lysozyme was used as a negative control antigen, and no discernible binding was demonstrated by any of the antibodies to this control. ATL5338 was used as a negative isotype control and showed no binding to HTT exon 1 48Q.

[0348] Table 23 shows the VH CDR sequences for each of the antibodies generated using combinations of mutations present in the affinity matured variants, along with the VH CDR sequences of ATL5895. The complete VH and VL sequences of these antibodies are shown in Table 1.

[0349] [Table 40]

[0350] Table 24 shows EC50 values ​​derived from sandwich ELISA for binding of the newly generated antibodies to exon 1 48Q HTT. All antibodies were shown to bind to HTT, except for the control antibody 5338 (isotype control, conjugated to fluorescein). EC50s were calculated using a variable slope (four parameter) nonlinear fit of the data. Each of the generated antibodies had similarly low EC50 levels. ATL_6375, 6376, and 6377 demonstrated the greatest binding affinity of the antibodies tested. The EC50 for 5895 is shown in Table 10 (2.077017E-09M). The EC50 for the comparative antibody ATL_0005059 / NI-302.8F1 is shown in Table 10 (1.725504 E-08 M).

[0351] [Table 41]

[0352] Example 18 –Mouse HTT ELISA To assess the binding potency of the antibodies to mouse HTT protein, ATL5895, ATL6376, and ATL6377 were evaluated in a direct ELISA format for binding to mouse HTT and human lysozyme (as negative control antigens). ATL5338 was used as a negative isotype control.

[0353] Figure 28 shows the results of indirect ELISA for antibody binding to mouse HTT. ATL5895, ATL6376, and ATL6377 all showed binding to mouse HTT, while ATL5338 did not bind to mouse HTT (isotype control). Lysozyme showed no appreciable binding to any of the antibodies evaluated at concentrations below 1 μM. These results indicate that ATL5895, ATL6376, and ATL6377 have cross-species reactivity with mouse HTT.

[0354] Example 19 – Binding of affinity matured antibodies by Biolayer Interferometry The binding interactions of ATL_5895 and affinity-matured antibodies described in Examples 11 and 17 above were assessed by biolayer interferometry (BLI) using an Octet instrument. Biotinylated peptides of HTT exon 1 (SEQ ID NO: 174) or mutant HTT exon 1 48Q (SEQ ID NO: 44) were loaded onto either streptavidin or GST biosensors (Sartorius, 18-5019 and 18-5096), respectively. To measure binding during this association phase, the sensors were subsequently immersed in separate wells containing the indicated concentrations of mAb for 300 seconds at 1000 rpm. Binding responses are reported at the end of the association phase for all mAbs.

[0355] Figure 29 shows the results of these experiments, which demonstrate that the affinity matured antibodies bind to HTT exon 1 and mutant HTT exon 1, and show increased binding propensity compared to the parental ATL_5895.

Claims

1. 1. An isolated antibody or antibody fragment thereof that specifically binds to a huntingtin (HTT) protein 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; 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 containing one or two substitutions compared to PPYYYYYGLDV (SEQ ID NO: 3), said substitutions being at positions selected from 95 and 97, said substitutions being selected from Y97F and P95S, and said 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. An isolated antibody or antibody fragment thereof, wherein LCDR3 has the amino acid sequence GSYAGTANV (SEQ ID NO: 6) or an amino acid sequence containing one, two, three or four amino acid substitutions compared to GSYAGTANV (SEQ ID NO: 6), said substitutions being selected from A92G, A95E, G89V and Y91F, and wherein the position numbering is Kabat.

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

3. The antibody (a) a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, 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 is a heavy chain variable (VH) domain having the amino acid sequence PPYYYYYGLDV (SEQ ID NO: 3); and a light chain variable (VL) domain, (1) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, 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 is a VL domain having the amino acid sequence GSYAGTANV (SEQ ID NO: 6); (2) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, 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 is a VL domain having the amino acid sequence VSYGGTENV (SEQ ID NO: 162); (3) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, 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 is a VL domain having the amino acid sequence VSFAGTANV (SEQ ID NO: 160); or (4) A VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, 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), a light chain variable (VL) domain selected from (b) a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3; 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 is a heavy chain variable (VH) domain having the amino acid sequence PPFYYYYGLDV (SEQ ID NO: 158); and a light chain variable (VL) domain comprising CDRs LCDR1, LCDR2 and LCDR3, 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 is a light chain variable (VL) domain comprising the amino acid sequence VSYGGTENV (SEQ ID NO: 162); or (c) a heavy chain variable (VH) domain comprising CDRs HCDR1, HCDR2 and HCDR3, 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 is a heavy chain variable (VH) domain having the amino acid sequence SPYYYYYGLDV (SEQ ID NO: 157); and a light chain variable (VL) domain, (1) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, 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 is a VL domain having the amino acid sequence VSYAGTANV (SEQ ID NO: 161); or (2) a VL domain comprising CDRs LCDR1, LCDR2, and LCDR3, 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), a light chain variable (VL) domain selected from 3. The isolated antibody or antibody fragment of claim 1 or 2, comprising:

4. the VH domain is a human VH domain, and / or the antibody or fragment thereof ATL_0005895 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCIP (SEQ ID NO: 172)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), ATL_0006376 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCVP (SEQ ID NO: 173)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), ATL_0006199 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYWCSP (SEQ ID NO: 169)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), and ATL_0006200 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVP (SEQ ID NO: 170)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), ATL_0006202 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98)-[CDRH1]-WVRQAPGKGLEWVG (SEQ ID NO: 99)-[CDRH2]-RFTISRDDSKNTLYLQMNSLKTEDTAVYYCSP (SEQ ID NO: 171)-[CDRH3]-WGQGTTVTVSS (SEQ ID NO: 101), or ATL_006195 VH framework sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFN (SEQ ID NO: 98) - [CDRH1] -WVRQAPGKGLEWVG (SEQ ID NO: 99) - [CDRH2] -RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTP (SEQ ID NO: 180) - [CDRH3] -WGQGTTVTVSS (SEQ ID NO: 101) 4. The isolated antibody or fragment thereof of claim 1, having a VH domain framework sequence selected from:

5. 5. The isolated antibody or fragment thereof of any one of claims 1 to 4, wherein the VL domain is a human VL domain and / or 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]-FGTGTKLTVL (SEQ ID NO: 139).

6. An isolated antibody or fragment thereof according to any one of claims 1 to 5, wherein HCDR1, HCDR2 and HCDR3 of the VH domain are in a germline framework, and / or LCDR1, LCDR2 and LCDR3 of the VL domain are in a germline framework.

7. the heavy chain variable domain comprises any of the amino acid sequences of ATL_5895 VH (SEQ ID NO: 7), ATL_6204 VH (SEQ ID NO: 7), ATL_6199 VH (SEQ ID NO: 144), ATL_6374 VH (SEQ ID NO: 148), ATL_6194 VH (SEQ ID NO: 145), ATL_6375 VH (SEQ ID NO: 145), ATL_6200 VH (SEQ ID NO: 145), ATL_6202 VH (SEQ ID NO: 146), ATL_6203 VH (SEQ ID NO: 147), ATL_6205 VH (SEQ ID NO: 148), ATL_6376 VH (SEQ ID NO: 175), ATL_6377 VH (SEQ ID NO: 176), ATL_6195 VH (SEQ ID NO: 179), or ATL_6378 VH (SEQ ID NO: 176); and 7. The isolated antibody or fragment thereof of any one of claims 1 to 6, wherein the light chain variable domain comprises any of the amino acid sequences of ATL_5895 VL (SEQ ID NO: 8), ATL_6199 VL (SEQ ID NO: 8), ATL_6195 VL (SEQ ID NO: 8), ATL_6002 VL (SEQ ID NO: 8), ATL_6374 VL (SEQ ID NO: 153), ATL_6375 (SEQ ID NO: 153), ATL_6376 VL (SEQ ID NO: 153), ATL_6378 VL (SEQ ID NO: 153), ATL_6194 VL (SEQ ID NO: 154), ATL_6377 VL (SEQ ID NO: 154), ATL_6203 VL (SEQ ID NO: 154), ATL_6204 VL (SEQ ID NO: 155), ATL_6205 VL (SEQ ID NO: 156), or ATL_6202 VL (SEQ ID NO: 159).

8. the heavy chain variable domain comprises the amino acid sequence of any of ATL_5895 VH (SEQ ID NO:7), ATL_6376 VH (SEQ ID NO:175), ATL_6377 VH (SEQ ID NO:176), or a sequence containing up to one, two, or three mutations compared to any of the foregoing sequences; and 8. The isolated antibody or fragment thereof of any one of claims 1 to 7, wherein the light chain variable domain comprises the amino acid sequence of any of ATL_5895 VL (SEQ ID NO: 8), ATL_6376 VL (SEQ ID NO: 153), ATL_6377 VL (SEQ ID NO: 154), or a sequence containing up to 1, 2, or 3 mutations compared to any of the foregoing sequences.

9. a. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_5895 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_5895 VL); or b. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 148) (ATL_6374 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6374 VL); or c. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6375 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6375 VL); or d. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPFYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 175) (ATL_6376 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6376 VL); or e. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 176) (ATL_6377 and the light chain variable domain sequence comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6377 VL); or f. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 176) (ATL_6378 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 153) (ATL_6378 VL); or g. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCSPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 144) (ATL_6199 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6199 VL); or h. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6200 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6002 VL); or i. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCSPPPFYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 146) (ATL_6202 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYGGTENVFGTGTKVTVL (SEQ ID NO: 159) (ATL_6202 VL); or j. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPSPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 147) (ATL_6203 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6203 VL); or k. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCIPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 7) (ATL_6204 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSFAGTANVFGTGTKVTVL (SEQ ID NO: 155) (ATL_6204 VL); or 1. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYWCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 148) (ATL_6205 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 156) (ATL_6205 VL); or n. The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 145) (ATL_6194 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCVSYAGTANVFGTGTKVTVL (SEQ ID NO: 154) (ATL_6194 VL); or The heavy chain variable domain has the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFNKAWMSWVRQAPGKGLEWVGRIKSGIDAGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTPPPYYYYYGLDVWGQGTTVTVSS (SEQ ID NO: 179) (ATL_6195 and the light chain variable domain comprises the amino acid sequence QSALTQPRSVSGSPGQSVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGATASLTISGLQAEDEADYYCGSYAGTANVFGTGTKVTVL (SEQ ID NO: 8) (ATL_6195 VL); or p. The isolated antibody or fragment thereof of any one of claims 1 to 8, wherein the heavy chain variable domain comprises a variable domain comprising an amino acid sequence that has at least 95% sequence identity or contains up to 1, 2, or 3 substitutions compared to any one of the heavy chain variable domains (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 contains up to 1, 2, 3, 4, or 5 substitutions compared to any one of the light chain variable domains (a) to (n).

10. the HTT protein is human or mouse HTT, and / or The isolated antibody or fragment thereof according to any one of claims 1 to 9, wherein the isolated antibody or fragment thereof binds to a region located within exon 1 of HTT.

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

12. The isolated antibody or fragment thereof of any one of claims 1 to 11, having higher relative binding to mutated and / or aggregated and non-mutated and / or non-aggregated HTT compared to a reference antibody (e.g., ATL_0005059, etc.).

13. The reference antibody a. 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) a heavy chain variable (VH) domain having b. 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) A light chain variable (VL) domain having 13. The isolated antibody or fragment thereof of claim 11 or 12, comprising: optionally, the reference antibody is NI-302.8F1 described in U.S. Pat. No. 11,401,325 B2.

14. 14. The isolated antibody or fragment thereof of any one of claims 1 to 13, which binds to mutant and / or aggregated HTT protein as determined by measuring immunoprecipitation of mutant HTT with said isolated antibody or fragment thereof, and optionally the mutant HTT is an HTT protein or fragment thereof comprising more than 35 glutamine residues in its polyQ tract, optionally a fragment comprising exon 1.

15. Recognizing an epitope in the region corresponding to exon 1 of the HTT gene, and / or recognizes an epitope located in the polyP region of HTT and / or recognizes an epitope comprising the amino acid sequence QQQQPPPPPPPPPPPPPP (SEQ ID NO: 47) or PQPQPPPPPPPPPPPPPP (SEQ ID NO: 48); and / or able to cross the blood-brain barrier, and / or 15. The isolated antibody or fragment thereof of any one of claims 1 to 14, which is a bispecific antibody further comprising a region that binds to a transferrin receptor.

16. 16. The isolated antibody or fragment thereof of any one of claims 1 to 15, which increases phagocytosis by a cell of an HTT protein or fragment thereof comprising exon 1 that comprises a 48Q repeat, optionally wherein the cell is a microglial cell, optionally an iPSC-derived human microglial cell, and optionally wherein the isolated antibody or fragment thereof increases phagocytosis by the cell of an HTT protein or fragment thereof comprising exon 1 that comprises a 48Q repeat in a dose-dependent manner, and / or wherein the increased phagocytosis is measured by detecting phagocytosis of beads coated with the HTT protein or fragment that are coated with a pH-sensitive fluorescent dye, and / or wherein the increased phagocytosis is measured as described herein (Materials and Methods).

17. 17. The isolated antibody or fragment thereof according to any one of claims 1 to 16, which reduces the aggregation rate of an HTT protein or a fragment thereof comprising exon 1 containing a 48Q repeat in a cell-free assay, and optionally said reduced aggregation rate is measured using a FRASE assay, and / or said reduced aggregation rate is measured as described herein (Materials and Methods), and / or immunodepletion of a solution comprising said HTT protein or a fragment thereof with said isolated antibody or fragment thereof results in a delta t50 of at most 0.1, at most 0.2, or at most 0.3 for aggregation of said HTT protein or fragment thereof, and / or said aggregation of said HTT protein or fragment thereof is measured in the presence of HTT fibrils and / or brain homogenates from one or more R6 / 2 mice.

18. 18. The isolated antibody or fragment thereof of any one of claims 1 to 17, which reduces the aggregation of mutant HTT, wherein the aggregation of said mutant HTT is measured as the presence and / or concentration of HTT aggregates in the brain of a transgenic mouse model of Huntington's disease, optionally an R6 / 1 mouse, and optionally, wherein treating said mouse with said isolated antibody or fragment thereof for 12 weeks or more results in a statistically significant reduction in the concentration of HTT aggregates in the striatum and / or cortex.

19. 19. The isolated antibody or fragment thereof of any one of claims 1 to 18, which binds to mutant HTT in vivo.

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

21. 21. The isolated antibody or fragment thereof of any one of claims 18 to 20, wherein the antibody or fragment thereof does not reduce the level of non-mutant 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-mutant and / or non-aggregated HTT is measured as the concentration of soluble and / or non-mutant HTT in the mouse, and optionally the mouse is an R6 / 1 mouse.

22. 22. The isolated antibody or fragment thereof of any one of claims 1 to 21, which maintains a monomer percentage of more than 95% or more than 97% after 4 weeks of incubation at -80°C, 4°C, 21°C, 40°C and / or after 10 freeze-thaw cycles, and / or binds to an HTT protein comprising exon 1 of HTT and the 48 glutamine residues in its polyQ tract as assessed by ELISA after 4 weeks of incubation at -80°C, 4°C, 21°C and 40°C and / or after 10 freeze-thaw cycles, 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 a set of DNA molecules encoding the antibody or antibody fragment thereof according to any one of claims 1 to 22.

24. 24. A vector or set of vectors encoding one or more DNA molecules according to claim 23.

25. 24. A host cell comprising the vector or set of vectors of claim 23.

26. 23. A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the isolated antibody or antibody fragment thereof of 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. 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-mutant and / or non-aggregated HTT in the subject.

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

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

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

31. 31. The method, use or composition for use of 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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