EA046697B1 - HUMAN ANTIBODIES TO HUNTINGTIN (HTT) AND THEIR USE - Google Patents

Description

Technical field

The present invention relates generally to antibody-based therapy for Huntington's disease (HD), which is associated with the huntingtin protein (HTP). In particular, the present invention relates to new molecules that specifically bind to human HTT and/or antigens thereof, in particular to human-derived antibodies, as well as HTT-binding fragments, synthetic and biotechnological derivatives of these antibodies, which can be used for treatment diseases and conditions induced by such HTT isoforms that cause the disease.

In addition, the present invention relates to pharmaceutical and diagnostic compositions containing such HTT-binding molecules, antibodies and their mimetics, which are valuable both as a diagnostic tool for identifying diseases and/or disorders associated with HTT aggregation, and for use in the strategy passive vaccination for the treatment of disorders associated with diseases associated with NTT amyloidosis.

State of the art

Huntington's disease (HD) is an autosomal dominant neurological amyloidogenic disorder. This autosomal disease affects 5 to 10 individuals in every 100,000 people. However, in the United States, the incidence of this disease is much higher; studies have shown that nearly 200,000 individuals in the United States have a 50% risk of developing HD; specifically, there are 30,000 patients registered in the United States, out of a total of 100,000 registered patients worldwide.

A number of studies have shown that HD develops as a result of expansion (elongation) of the CAG trinucleotide repeat in the huntingtin gene (HTT), in particular, in exon 1 of the HTT gene, located on chromosome 4 (MacDonald et al., Cell 72, (1993), 971 -983), which leads to the formation of an extended region (tract) of polyglutamine (poly-Q) in the HTT protein as a result of translation. HD occurs if the length of the polyglutamine tract exceeds a threshold of 35-40 glutamine residues, and there is a strong inverse correlation between the length of the repeat region and the age at which the disease manifests. This poly-Q region causes misfolding and aggregation of HTT in several areas of the body, for example, in neurons and glial cells. With age, NTT aggregates accumulate, which leads to degeneration of GABAergic striatal neurons and cortical pyramidal neurons. Symptoms of ITT misfolding and aggregation include involuntary movements, incoordination, depression, cognitive decline such as memory loss and/or dementia.

Since 1993, when the mutation causing HD was identified, the understanding of the pathophysiology and molecular biology of this disease has improved significantly. Medications such as Xenazine® (tetrabenazine, Lundbeck), a VMAT2-inhibiting hexahydrodimethoxybenzoquinolysine derivative, have been developed for symptomatic treatment of involuntary muscle movements.

In addition, gene silencing approaches such as RNA interference (RNAi) have been proposed as potential therapies. In particular, the use of siRNA against the HTT gene in a mouse model of HD (R6/2) has been shown to inhibit the expression of the mutant HTT gene, see, for example, references: Warby et al., Am. J. Hum Genet. 84 (2009), 351-366 and Olshina et al., Biological Chemistry 285 (2010), 21807-21816. However, one of the limitations of this method is the difficulty of introducing a sufficient amount of siRNA into target cells or tissues, as shown, for example, by Boudreau et al. (Brain Research 1338 (2010), 112-121). In addition, there may be a need to ensure the safety of these approaches, since gene deletion studies in animal models and cultured cells suggest the need for continuous expression of huntingtin (Dragatsis et al., Nat. Genet. 26 (2000), 300 -306; Gauthier et al., Cell. 118 (2004), 127-138; Zuccato et al., 35 (2003), 76-83).

Accordingly, there is a need for new therapeutic strategies for effective and safe treatment of diseases associated with HTT aggregation, preferably directly affecting amyloid formation by mutant HTT.

This technical problem is solved by embodiments of the present invention described in the claims and further described below and illustrated with examples and drawings.

Brief description of the invention

The present invention provides anti-huntingtin antibodies (HTT) and equivalent HTT-binding molecules for use in the prophylactic or therapeutic treatment of diseases and conditions associated with HTT amyloidosis. More specifically, therapeutically useful human-derived antibodies are provided that recognize mutated and/or aggregated forms of HTT, as well as HTT-binding fragments, synthetic and biotechnological derivatives of these antibodies.

In particular, in the course of experiments carried out in accordance with the present invention, recombinant cloning and production of human-derived monoclons were successfully carried out.

- 1 046697 natural HTT-specific antibodies, specific for mutated and/or aggregated types of HTT, and/or their fragments. The human subjects from whom the B Cells from which the cDNA encoding the variable domain of the human-derived monoclonal antibody to HTT was isolated were respectively healthy donors. However, in another embodiment of the present invention, B cells from which human-derived monoclonal antibodies to HTT and the cDNAs encoding their variable domain, respectively, can be isolated are obtained from HD patients who have had a CAG trinucleotide repeat expansion in the HTT gene, who are asymptomatic or demonstrate unusually slow or stable disease progression; or, alternatively, demonstrating typical clinical signs of Huntington's disease. Additionally, as exemplified, the antibodies of the present invention are capable of attenuating dendritic spine loss, improving behavioral performance during task-directed learning, and enhancing sensorimotor abilities in a mouse model of HD. Accordingly, it is reasonable to expect that the human anti-HTT monoclonal antibodies of the present invention and derivatives thereof, in addition to being non-immunogenic, will also provide beneficial therapeutic effects in humans.

According to the present description, in the Background Art section, it is described that to date, attempts have been made to block the pathogenesis of HD using intracellular methods such as RNA interference (RNAi); see also, for example, Stanek et al., Human Gene Therapy 25 (2014), 461-474, which describes silencing of mutant huntingtin using adeno-associated virus-mediated RNA interference. Regarding the immunotherapeutic approach, the intracellular expression of single chain antibody fragments (scFv), i.e., has been investigated over the last decade. intrabodies lacking an immunoglobulin constant region, for example, the IgG class; see, for example, above and Butler et al., Prog Neurobiol. 97 (2012), for engineered intracellular scFv and single domain (dAb; nanobody) antibody therapy to counteract mutant huntingtin and related toxic intracellular proteins.

For example, in the source: Lecerf et al., Proc. Nat. Acad. Sci. 98 (2001), 4764-4769 describes an antibody consisting of a single chain fragment variable region (scFv) specific for the 17 N-terminal residues of huntingtin adjacent to the polyglutamine encoded by exon 1 in HD, selected from a large human phage display library. The corresponding scFv antibody, scFv-C4, containing the lambda light chain variable region (V _L ) (Kvam et al., PLoS One 4 (2009), e5727; GenBank accession number ACA53373) is reported to have some neuroprotective effects in transgenic mice B6.CgHDR6/1 in a mouse model of HD, however, this effect weakens both with greater severity of the disease and also over time after administration. To improve intrabody steady-state concentrations and to target the N-terminal protein fragments encoded by htt exon 1 (httex1) bound by scFv-C4 to the proteasome for degradation to prevent their aggregation, the mouse ornithine decarboxylase PEST signal sequence was appended to the scFv-C4 antibody ( mODC); see Butler and Messer, PLoS One 6 (2011), e29199. In vivo experiments have not yet been reported.

Also, the group of Khoshnan et al. was engaged in the development of therapeutic agents based on intrabodies to HD and, among other things, they described scFv antibodies against huntingtin, derived from mouse monoclonal antibodies, binding epitopes of polyglutamine (poly-Q), polyproline (poly-P ) and End, and their effects on the intracellular expression, aggregation and toxicity of mutant huntingtin; see, for example, Ko et al., Brain Research 56 (2001), 319-329, Khoshnan et al., Proc. Nat. Acad. Sci 99 (2002), 1002-1007 and Legleiter et al., J. Biol. Chem. 284 (2009), 21647-21658 and their US patent application 2003/0232052 AI. The US patent application also describes a human scFv antibody called hMW9, which was isolated from a human scFv phage library using a recombinant mutant huntingtin protein. However, unlike the mouse monoclonal antibody-derived scFvs MW1, MW2, MW7, and MW8, no sequence data for hMW9 was provided; it has also not been reported again to date.

Colby et al., Proc. Nat. Acad. Sci. 342 (2004), 901-912 describes the development of a human intracellular antibody light chain variable domain (V _L ) specific for the amino terminus of huntingtin using a yeast surface display-based non-immune human antibody library. This single-domain intrabody, consisting only of the lambda light chain domain of the parent scFv, has been reported to inhibit huntingtin aggregation in a cell-free in vitro assay as well as in a mammalian cell culture model of HD; see also the corresponding international application WO 2005/052002. In this case, in vivo experiments have also not yet been reported.

Thus, it is clear that existing intrabody-based approaches are either only suitable for cell-based analysis or their successful application in animal models of HD, at least in long-term experiments, has not yet been confirmed. In particular, intrabodies exhibit a number of limitations in vivo, such as their potential toxicity due to intracellular/intranuclear accumulation of intrabody-antigen complexes, or limited distribution, e.g.

- 2 046697 delivery to areas of the human brain that have a significant volume; see, for example, Butler et al., Prog. Neurobiol. 97 (2012), 190-204 and Sothwell et al., J. Neurosci. 29 (2009), 13589-13602. In addition, a general disadvantage of intracellular methods is the problem of targeting the antibody and the DNA vector encoding it, respectively, to the desired cells; and, when delivered exogenously, an inconvenient administration schedule, such as intrastriatal injections; see, for example, Snyder-Keller et al., Neuropathol. Exp. Neurol. 69 (2010), 1078-1085. In addition, general problems associated with gene therapy and the use of viral vectors remain.

At the same time, experiments carried out in accordance with the present invention showed for the first time that full-length IgG antibodies against various epitopes of huntingtin, when administered systemically, can be successfully delivered to the brain (Example 24 and Fig. 18), and that antibodies according to the present The invention is capable of attenuating the loss of dendritic spines, improving behavioral performance during goal-directed learning to perform specific tasks, and increasing sensorimotor abilities in a mouse model of HD (Example 34 and FIG. 34).

Accordingly, as shown in the Examples section, an anti-HTT antibody or an HTT-binding fragment, synthetic or biotechnological derivative of said antibody is preferably of the IgG class, that is, it contains, as is generally known and described herein, two identical variable region heavy (VH) polypeptides. ) chain and two identical light chain variable region (VL) polypeptides, as well as a constant region and a domain, respectively, i.e. at least one or all of the light chain (CL) and heavy chain (CH1, CH2 or CH3) constant domains. In other words, according to one aspect of the present invention, there are provided recombinantly expressed bivalent antibodies specific for huntingtin and its aggregated forms, fragments, peptides and derivatives, suitable for use in the treatment or in vivo diagnosis of huntingtin and related disorders, and characterized by the presence of a constant immunoglobulin areas. Additionally, as described below herein, the immunoglobulin may be of any class, such as IgG, IgM, IgA, IgG, or IgE, and corresponding immunoglobulin subclasses (isotypes), such as lgG1, lgG2, lgG3, lgG4, lgA1. Preferably, however, said antibody is of the human IgG subtype.

Additionally, as described herein, the human-derived antibodies of the present invention are characterized by the presence of at least one or more human-derived CDR regions, i.e. encoded by cDNAs derived from human memory B cells; wherein preferably the VH and/or VL chain is also derived from human memory B cells. The constant region or any domain thereof, if derived from a human, may be derived from the same source as the CDR region(s) and the VH and/or VL chain, respectively, or from a different source. source.

As used herein, unless otherwise indicated or obvious from the context, antibodies of the present invention referred to herein include the human-derived antibodies illustrated in the examples, as well as HTT-binding fragments, synthetic and biotechnological derivatives thereof.

Additionally, it may be noted that according to the prior art related to the production of intrabodies derived from a human scFv phage library, scFcv was almost always obtained from the V-lambda variable light chain; see Kvam et al. and Colby et al., supra. Moreover, more than 90% of the human-derived antibodies of the present invention use the V-kappa light chain, including antibodies NI-302.31F11 and NI-302.35C1, according to example 24, capable of penetrating the brain when administered systemically, and, according to example 34 (NI-302.35C1), which has beneficial effects on behavioral activity and performance-related tasks in mice in an animal model of HD. Accordingly, it is hypothesized that antibodies containing a V-kappa light chain may have superior performance compared to antibodies containing a V-lambda-derived light chain. Accordingly, in a preferred embodiment, the antibodies of the present invention comprise a variable light chain derived from V-kappa.

As shown in the examples section and the drawings, the anti-HTT antibody, its HTT-binding fragment, synthetic and biotechnological variant bind to different regions of the HTT protein encoded by exon 1, which exhibits a toxic change as described above, i.e. elongation due to unstable trinucleotide repeats, as shown in the examples section. Specifically, the antibody of the present invention recognizes the polyproline region, polyglutamine/polyproline region, proline-rich region, C-terminal region, or N-terminal region of the HTT protein encoded by exon 1. The epitopes of the subject antibodies presented in the examples section are summarized in FIG. 20. As mentioned in the Background Art section, GD occurs when the length of the polyglutamine tract exceeds a threshold of 35-40 glutamine residues due to HTT aggregation. Accordingly, as shown in Example 3, aggregated and soluble Htt proteins encoded by exon 1 with 21, 35 or 49 polyglutamine repeats were prepared and tested for binding with the identified antibodies. Below, these structures will be designated as HDX, when

- 3 046697 This X denotes the number of glutamine residues, for example, exon 1 of HTT with 21 polyglutamine repeats would be designated HD21. Accordingly, unless specifically stated otherwise, the term HTT refers to HTT encoded by exon 1, and soluble HTT refers to the corresponding GST fusion proteins.

According to a preferred embodiment of the present invention, said anti-HTT antibody or HTT binding fragment, synthetic or biotechnological derivative of said antibody is capable of preferentially binding aggregated forms of HTT or misfolded forms of HTT. As described, for example, in the source: Legleiter et al., JBC 285 (19) (2010), 14777-14790 and in the examples section, the aggregation of HDX proteins, in terms of speed and size, increases with the number of glutamine residues (Q).

According to a particular preferred embodiment of the present invention, an anti-HTT antibody or an HTT-binding fragment thereof, a synthetic or biotechnological derivative thereof exhibits the immunological binding characteristics of an antibody characterized by the presence of any of the VH and/or _VL variable regions of FIG. 1. Preferably, the variable region of the antibody contains at least one complementarity determining region (CDR) of the VH and/or _VL variable regions, i.e. pairs of circuits VH and V _L according to Fig. 1A-1AU, with one or more amino acid substitutions possible as long as the resulting antibody retains similar binding specificity as the antibody in question containing the corresponding pair of VH and VL chains according to FIG. 1A-1AU, as shown in the examples section, for example, according to the general information in FIG. 20, i.e. epitope specificity is maintained and EC ₅₀ values are maintained in the same order of magnitude for a particular antigen, preferably within at least a 50%, more preferably at least 25%, and most preferably within at least a 10% range of values . Preferably, one, two, or all three CDR regions of the VH and VL chain contain at least one conserved amino acid at corresponding positions (i.e., an identical amino acid or a conservative substitution) for at least about 20%, preferably for about 40%, more preferably for about 50% and most preferably for about 75% of the VH and VL chain amino acid sequences, respectively, of the subject antibodies that recognize the same type of HTT epitope, i.e. polyproline region, proline-rich region, C-terminus or N-terminus. For example, sequence alignments of the antibodies in question show the presence of predominantly one or two tyrosines (Y) in CDRH1; see fig. 36. A similar conserved amino acid can also be identified in other CDRs.

According to a further embodiment of the present invention, an anti-HTT antibody or an HTT-binding fragment thereof, a synthetic or biotechnological derivative of the antibody is a bispecific antibody. Accordingly, an antibody of the present invention may be capable of recognizing at least two different epitopes on the same or different antigens. For example, the first antigen binding site, i.e. the variable domain may be specific for HTT and preferably contains the variable region of any of the subject antibodies presented in the accompanying examples and drawings, and the second antigen binding site may be specific for another, preferably also neurotoxic protein, and contain the variable region of the corresponding antibody . Thus, protein misfolding and aggregation represents a major hallmark of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and HD. Although until recently the general consensus was that each aggregation-prone protein was characteristic of a specific disorder [α-synuclein (α-syn) for PD, mutant huntingtin (HTT) for HD, Tai and beta-amyloid peptide for AD], all more evidence indicates that aggregation-prone proteins are in fact capable of coaggregation and mutual modification of behavior and toxicity, so that this process may also presumably contribute to the occurrence of overlapping clinical symptoms in different diseases; see, for example, co-aggregation of α-syn and mutant HTT by Pocas et al., Hum. Mol. Genet. 24 (2015), 1898-1907.

Accordingly, according to one embodiment of the present invention, an anti-HTT antibody or an HTT-binding fragment thereof, a synthetic or biotechnological derivative of the antibody is a bispecific antibody capable of binding HTT and a protein associated with a neurodegenerative disorder, particularly in the brain, preferably selected from the group consisting of α-synuclein, Tai, beta-amyloid peptide, SOD1, C9orf72 and TDP-43; see, for example, Blokhuis et al., Acta Neuropathol. 125 (2013), 777-794. Human-derived monoclonal antibodies to these proteins are known in the art; see, for example, international application WO 2008/081008, which describes an antibody to A-beta, WO 2010/069603, which describes an antibody to asynuclein; WO 2012/049570, which describes an antibody to tau; WO 2012/080518, which describes an antibody to SOD1; WO 2012/113775, which describes an antibody to ankyrin; WO 2013/061163, which describes an antibody to TDP-43; and European patent application EP 14187180.6 and subsequent international application, where

- 4 046697 an antibody to C9orf72 is described. Bispecific and multispecific antibodies can be produced using methods well known in the art, for example, by chemical recombination of monoclonal immunoglobulin G1 fragments as described, for example, in Brennan et al., Science. 229 (1985), 81-83, or simultaneous recombinant co-expression of suitable heavy and light chains and appropriate pairing; see, for example, Lewis et al., Nature Biotechnology 32 (2014), 191-198; For reviews, see, for example, Kontermann, mAbs4 (2012), 182-197 and Kontermann and Brinkmann, Drug Discovery Today 20 (2015), 838-847.

In an alternative or additional embodiment, the bi- or multispecific antibody comprises at least first and second antigen binding sites, i.e. a variable domain specific for two distinct HTT epitopes, wherein preferably one or both variable regions are derived from any of the subject antibodies presented in the accompanying examples and drawings and further described herein. Accordingly, in a preferred embodiment, the bispecific antibody of the present invention contains two antibody binding sites/domains that recognize a polyproline region, a polyglutamine/polyproline region, a proline-rich region, a C-terminal region, an N-terminal region, or a conformational epitope of an exon-encoded HTT protein 1. The epitopes of the exemplified antibodies in question are summarized in FIG. 20. Accordingly, in one embodiment, the bispecific antibody of the present invention recognizes at least two different epitopes shown in FIG. 20, and is distinguished by the combined binding characteristics of the cognate antibody in question, respectively.

The antigen binding fragment of any of the antibodies contemplated herein may be a single chain Fv fragment, an F(ab') fragment, an F(ab) fragment and an F(ab') ₂ fragment, or any other antigen binding fragment. Moreover, as mentioned, according to a specific preferred embodiment, said antibody or an HTT-binding fragment, synthetic or biotechnological derivative of said antibody is a human antibody of the IgG isotype and contains at least part of a constant region. Alternatively, the antibody is a hybrid (chimeric) human-rodent antibody; or an antibody containing rodent antibody elements, such as a mouse antibody or containing mouse antibody elements; rat antibody or containing elements of rat antibodies; Antibody variants originating from rodents are particularly suitable for use in diagnostic methods and for animal research.

In addition, the present invention relates to compositions containing an antibody according to the present invention or an antigen-binding fragment thereof, a synthetic or biotechnological derivative of the specified antibody, as well as methods of immunotherapy and immunodiagnostics using such compositions for the prevention, diagnosis or treatment of diseases and/or disorders associated with NTT amyloidosis, characterized in that an effective amount of said composition is administered to the patient in need thereof.

The present invention also relates to polynucleotides encoding at least the variable region of an immunoglobulin chain of an antibody according to the present invention. Preferably, said variable region comprises at least one complementarity determining region (CDR) VH and/or _VL variable region according to FIG. 1. Preferably, said polynucleotide is cDNA.

Accordingly, the present invention also covers vectors containing these polynucleotides and host cells transformed therewith, as well as their use for the production of antibodies and equivalent binding molecules specific for HTT and preferably capable of binding mutated and/or aggregated HTT species or fragments thereof. Methods and methods for recombinantly producing antibodies and their mimics, as well as methods for screening for competitive binding molecules that may or may not be antibodies, are known in the art. However, as described herein, in particular for therapeutic use in humans, the antibody of the present invention is a human antibody in the sense that the use of said antibody does not substantially induce an immune response directed against such antibody as is observed with the use of hybrid and even humanized antibodies. Thus, the present invention also relates to the use of cDNA, vector and host cell described herein and illustrated in the examples section for the production of an anti-HTT antibody, in particular a human-derived anti-HTT antibody or a biotechnological derivative thereof.

In addition, compositions and methods as described herein are provided that can be used to identify HTT, particularly mutated and/or aggregated HTT species or fragments in vitro, eg, in samples and/or in vivo. The described anti-HTT antibodies and their binding fragments can be used to screen human blood, plasma, serum, saliva, peritoneal fluid, cerebrospinal fluid (CSF) and urine for the presence of NTT and/or mutated and/or aggregated HTT species or fragments thereof in samples. for example, using

- 5 046697 ELISA-based assay or adapted surface assay. In one embodiment, the present invention relates to a method for diagnosing or monitoring the progression (control) of a disease and/or disorder associated with mutated and/or aggregated HTT species or fragments thereof in a subject, the method comprising determining the presence of mutated and/or aggregated HTT species or fragments thereof. HTT species or fragments in a sample from a subject being diagnosed using at least one antibody of the present invention, or an HTT binding molecule, and/or binding molecules for mutated and/or aggregated HTT species or fragments having substantially the same binding specificity as any of the above, with the presence of mutated and/or aggregated HTT species or fragments indicating the presence of the disorder.

Accordingly, the present invention also relates to a method for preparing a pharmaceutical composition for use in the treatment of a disorder associated with or caused by HTT aggregates, the method comprising:

(a) expressing a cDNA according to the present invention and/or culturing a host cell according to the present invention under culture conditions suitable for the production of an anti-HTT antibody, in particular a human-derived anti-HTT antibody or a biotechnological derivative thereof;

(b) purifying said antibody, biotechnological derivative thereof, or immunoglobulin chain(s) from the reaction mixture and culture, respectively, to pharmaceutical grade purity; and (c) admixing said antibody or biotechnological derivative thereof with a pharmaceutically acceptable carrier.

In addition, according to one embodiment of the present invention, there are provided anti-HTT antibodies and HTT-binding molecules comprising at least one CDR region of the antibody of the present invention to form a composition for in vivo detection (also referred to as in vivo imaging) or targeting therapeutic and/or or a diagnostic agent for NTT, in particular mutated and/or aggregated species or fragments of NTT in humans or animals. The methods and compositions described herein may be useful in diseases and/or disorders associated with HTT aggregation or amyloidosis and characterized, for example, by the occurrence of aggregated forms of HTT; they can be used to monitor the progression of disease and the therapeutic effectiveness of the applied therapy in a subject, for example, in in vivo imaging-related diagnostic methods. In one embodiment, said in vivo detection (imaging) includes scintigraphy, positron emission tomography (PET), single photon emission tomography (SPET), near-infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).

Thus, it is an object of the present invention, in particular, to provide methods for treating, diagnosing or preventing a disease and/or disorder associated with NTT amyloidosis. These methods involve administering to a subject, preferably a human antibody or a human antibody derivative at an effective concentration, wherein said antibody targets HTT or fragments thereof, preferably mutated and/or aggregated HTT species or misfolded HTT species, or fragments thereof.

According to a further aspect, the present invention provides a peptide containing an HTT epitope, preferably a mutated and/or aggregated species of HTT or fragments thereof, specifically recognized by an antibody of the present invention. Said peptide contains or consists of the amino acid sequence set forth below in the detailed description and examples, or a modified sequence thereof where one or more amino acids are replaced, deleted and/or added. In addition, the present invention provides a method for diagnosing diseases and/or disorders associated with NTT amyloidosis in a subject, comprising the step of determining the presence of an antibody that binds to the specified peptide in a biological sample obtained from the specified subject.

Additional embodiments of the present invention will be apparent from the following description and examples.

Fig. 1: Amino acid sequences of the variable regions of human antibodies NI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31F11, NI-302.2A2, NI-302.6N9, NI-302.74C11, NI-302.15F9, NI302. 39G12, No.-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, NI302.78H12, NI-302.71F6, No. 302.11N6, NI-302.3D8, NII-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9, NI-3O2.15E8, NI-302.15D3, NI-302.64E5, NI-302.7D8, NI-302.72F10, NI-302.12H2 , NI-3O2.8M1 and NI302.4A6. Framework (FR) and complementarity determining regions (CDR) are marked; CDR areas are underlined. The Kabat numbering system was used (see https://www.bioinf.org.uk/abs/).

Fig. 2: Characterization of huntingtin proteins and aggregates (HTT) encoded by exon 1. (A) Cloning of expression constructs GST-HttEx1Q21 (GST-HD21), GST-HttEx1Q35 (GSTHD35) and GST-HttEx1Q49 (GST-HD49); (B) Coomassie SDS-PAGE staining of only purified GST (lane 1), GST-HttExon1-Q21 (GST-HD21, lane 2), GST-HttExon1-Q35 proteins

- 6 046697 (GST-HD35, lane 3) and GST-HttExon1-Q49 (GST-HD49, lane 4), showing high purity, but also several additional bands; (C) Time-resolved characterization of in vitro aggregation reaction products of HD21, HD35, and HD49 using dot blot (left) and filter delay analysis (right) with polyclonal antibody HD-1 as detector antibody. HD35 aggregation reactions at 24 hours or HD49 reactions at 3 hours show aggregates larger than the 0.2 µm pore size detected by HD-1 in the filter delay assay; (D) In vitro determination of HD35 and HD49 composition using electron microscopy. HD35 aggregation reactions after 24 h [A, E] or HD49 reactions after 1 h [B, F], 3 h [C, G] or 24 h [D, H]. General view [A-D] with a magnification of 1000x and detailed structures [E-H] with a magnification of 66,000x.

Fig. 3: Determination of binding affinity of antibody NI-302.33C11 to the polyproline domain. (A) binding affinity of NI-302.33C11 for different HTT species determined using direct ELISA; (B) EC ₅₀ determination of NI-302.33C11 for aggregated HD49 (·), aggregated HD21 (), soluble GST-HD49 (a), and GST-HD21 (▼) exon 1-encoded Htt proteins using direct ELISA. Antibody NI-302.33W1 binds all four HTT species with similar EC ₅₀ values and (C) Time-resolved dot blot analysis of NI-302.33C11 binding to HTT aggregates in HD21, HD35 and HD49 aggregation reactions (left) and filter delay assays (right), with preferential late reaction binding of (aggregated) HD35 and HD49 in dot blot assays and HD35 and HD49 aggregates in filter delay assays.

Fig. 4: Determination of the binding epitope of antibody NI-302.33C11 by scanning overlapping peptides. Top: Pepscan image after hybridization of antibody NI-302.33C11. Bottom: General images of peptide sequences bound by antibody NI-302.33C11. Overlapping amino acids in the peptides (putative binding epitope) recognized by antibody NI302.33C11 are highlighted in bold in the consensus sequences. The HRP-conjugated donkey anti-human IgG Fcy detector antibody does not by itself bind any of the linear huntingtin peptides.

Fig. 5: NI-302.33C11 binds to the polyproline domain of HTT. Determination of EC ₅₀ for GSTHD49 (·), BSA-conjugated proline-rich peptide domain (♦), BSA-conjugated C-terminal peptide (), or BSA-conjugated polyproline peptide (A) using direct ELISA.

Fig. 6: Determination of the purity and integrity and binding specificity of the NI302.33C11 antibody. SDS-PAGE analysis followed by Coomassie staining with 2 and 10 μg of recombinant human antibody NI-302.33C11 to the polyproline domain.

Fig. 7: Determination of antibody binding affinity to the proline-rich domain of NI302.63F3.

(A) Binding affinity of NI-302.63F3 for different HTT species determined using direct ELISA; (B) Determination of EC50 NI-302.63F3 for aggregated HD49 (·), aggregated HD21 (), soluble GST-HD49 (A), and GST-HD21 (▼) exon 1-encoded Htt proteins using direct ELISA. Antibody NI-302.63F3 has similar EC ₅₀ values for all four species; (C) Time-resolved in vitro characterization of antibody NI-302.63F3 for the in vitro aggregation products of HD21, HD35, and HD49 using dot blot (left) and filter delay analysis (right) with preferential binding to huntingtin. extended by poly-Q tracts (HD49>HD35) in dot blot assays, and HD35 and HD49 aggregates in filter delay assays.

Fig. 8: Determination of the binding epitope of antibody NI-302.63F3 by scanning overlapping peptides. Top: Pepscan image after hybridization of antibody NI-302.63F3. Bottom: General images of peptide sequences bound by antibody NI-302.63F3. Overlapping amino acids in different peptides (putative binding epitope) recognized by the NI302.63F3 antibody are highlighted in bold in the consensus sequences. The HRP-conjugated donkey anti-human IgG Fcy detector antibody does not by itself bind any of the linear huntingtin peptides.

Fig. 9: NI-302.63F3 binds to the proline-rich HTT domain. Determination of EC ₅₀ for GSTHD49 (·), BSA-conjugated proline-rich peptide domain (♦), BSA-conjugated C-terminal peptide (), or BSA-conjugated polyproline peptide (A) using direct ELISA.

Fig. 10: Determination of the purity and integrity, and binding specificity of the NI302.63F3 antibody. SDS-PAGE analysis followed by Coomassie staining with 2 and 10 μg of recombinant human antibody NI-302.63F3 to the proline-rich domain.

Fig. 11: Determination of antibody binding affinity to the C-terminal domain of NI 302.35C1. (A) Binding affinity of NI-302.35C1 for different HTT species determined using direct ELISA; (B) determination of EC ₅₀ NI-302.35C1 for aggregated HD49 (·), aggregated HD21 (), soluble GST-HD49 (A) and GST-HD21 (▼) Htt proteins encoded by exon 1, using direct ELISA; (C) Time-resolved in vitro characterization of antibody NI-302.35C1 for the in vitro aggregation reaction products of HD21, HD35, and HD49 using dot blot (left) and filter delay analysis (right), with preferential binding to late (aggregated) ) reaction products of HD35 and HD49 in the dot blot analysis, and of HD35 and HD49 aggregates in the filter delay analysis.

Fig. 12: NI-302.35C1 binds to the BSA-conjugated C-terminal peptide domain of HTT. Determination of EC50 for GST-HD49 (·), BSA-conjugated proline-rich peptide domain (♦), BSA-conjugated C-terminal peptide (), or BSA-conjugated polyproline peptide (^) using direct ELISA.

Fig. 13: Determination of the purity and integrity, and binding specificity of the NI302.35C1 antibody. SDS-PAGE analysis followed by Coomassie staining with 2 and 10 μg of recombinant human antibody NI-302 to the C-terminal domain.

Fig. 14: Determination of antibody binding affinity to the N-terminal domain of NI302.15E8.

(A) Binding affinity of NI-3O2.15E8 for different HTT species determined using direct ELISA; (B) EU ₅₀ definition NI-302.15E8 for aggregated HD49 (·), aggregated HD21 (). soluble GST-HD49 (A) and GST-HD21 (▼) Htt proteins encoded by exon 1 using direct ELISA. Antibody NI-3O2.15E8 has higher EC ₅ o values for binding affinity to non-aggregated species.

Fig. 15: NI-302.15E8 binds to the BSA-conjugated N-terminal peptide domain of HTT. EC ₅₀ determination for GST-HD49 (·), BSA-conjugated N-terminal peptide (▼), BSA-conjugated proline-rich domain peptide (♦), BSA-conjugated C-terminal peptide (), or BSA-conjugated polyproline peptide (A ) using direct ELISA.

Fig. 16: Target specificity assay using direct ELISA. NI-302 antibodies (A) NI-302.33C11, (B) NI-302.63F3 and (C) NI-302.35C1, and (D) NI-3O2.15E8 do not bind irrelevant aggregating protein targets as shown in the binding specificity assay using direct ELISA.

Fig. 17: Spine density is significantly reduced in hippocampal slice cultures from Tg(HDexon1)62Gpb/1J transgenic mice compared with nontransgenic littermates. (A-D) Comparison of gross appearance of GFP-positive hippocampal neurons from nontransgenic littermates (A, C) and Tg(HDexon1)62Gpb/1J mice (B, D), and a single dendrite with individual spines at higher magnification (C, D). (E) Significant reduction in dendritic spine density in transgenic animals compared to wild-type animals (n=3-7 sections per group consisting of 2 wild-type animals or 3 transgenic animals). (F) Reduction in loss of dendritic spine density with antibodies NI-302.11F11 and 302.63F3 in sections from transgenic mice. (n=8-13 sections per group, including a total of 12 transgenic animals). Data shown are mean ± SEM. *p<0.05 (MWU), # p=0.05.

Fig. 18: Penetration of NI-302 antibodies into the brain in the R6/1 animal model. (A) Average drug levels of NI-302.31F11 (·) and NI-302.35W () in plasma and brain of R6/1 transgenic animals after a single intraperitoneal injection at a dose of 50 mg/kg. Data shown are mean ± SEM. n=3 for each group; (B) Drug levels in plasma and brain of individual mice following a single dose of 50 mg/kg.

Fig. 19: Determination of EC ₅₀ human-derived HTT antibodies for aggregated HD49 (·), aggregated HD21 (), soluble GST-HD49 (A) and GST-HD21 (▼) exon 1-encoded Htt proteins using direct ELISA . Some antibodies (eg NI-302.37C12 (I), NI302.55D8 (J), NI-3O2.11A4 (F) or NI-302.22H9 (G)) appear to bind primarily to the non-truncated GST-HTT protein , which indicates the preferential recognition of constructs with non-truncated soluble GST-BG by these antibodies, while some antibodies (for example, NI-302.74C11 (C) or NI-302.71F6 (M)) demonstrated high binding affinity, with similar EC values, with all formulations with HTT, suggesting that they bind to an epitope similar to that displayed on aggregated non-truncated exon 1 HTT ELISA constructs.

Fig. 20: Determination of binding affinity using direct ELISA. Affinity of human-derived HTT-specific antibodies for binding of different HTT proteins

Fig. 21: In vitro time-resolved characterization of antibodies NI-302.44D7, NI-302.37C12, NI-302.15F9 and NI302.71F6 for in vitro aggregation products HD21, HD35 and HD49 using dot blot (left) and analysis filter delay method (right), with preferential binding in particular of NI-302.15F9 and NI-302.71F6 to the late (aggregated) reaction products HD35 and HD49 in dot blot assays, and to the SDS-stable aggregates HD35 and HD49 in filter delay analysis.

- 8 046697

Fig. 22: Target specificity assay using direct ELISA. Antibodies NI-302 (A) NI-302.31F11, (B) NI-302.6N9, (C) NI-302.46C9, (D) NI-302.8F1, (E) NI-302.2A2, (F) NI302.74C11 , (G) NI-302.15F9, (H) NI-302.39G12, (I) NI-302.11A4, (J) NI-302.22H9, (K) NI-302.44D7, (L) NI302.55D8, (M ) NI-302.7A8, (N) NI-302.78H12, (O) NI-302.71F6, (P) NI-302.11H6, and (Q) NI-302.3D8 do not bind irrelevant aggregating protein targets as shown in the assay for binding specificity using direct ELISA

Fig. 23: Determination of the binding epitope of the NI-302 antibody by scanning overlapping peptides. Top: Pepscan image after hybridization of antibody NI-302. Bottom: General images of peptide sequences and binding scores of the NI-302 antibody to single peptides are shown. Overlapping amino acids in different peptides (putative binding epitope) recognized by the NI-302 antibody are highlighted in gray in the consensus sequences. The HRP-conjugated donkey anti-human IgG Fcy detector antibody does not by itself bind any of the linear huntingtin peptides. (A) 1 μg/ml NI-302.31F11 on a 21-spot membrane, (B) 1 μg/ml NI-302.74C11 on a 16-spot membrane, (C) 1 μg/ml NI-302.15F9 on a 16-spot membrane , (D) 1 μg/ml NI-302.39G12 on a 16-spot membrane, (E) 1 μg/ml NI-3O2.11A4 on a 16-spot membrane, (F) 1 μg/ml NI-302.22H9 on a membrane with 16 spots, (G) 1 µg/ml NI-302.44D7 on 16 spot membrane, (H) 1 µg/ml NI-302.37C12 on 16 spot membrane, (I) 1 µg/ml NI-302.55D8 on membrane with 16 spots, (J) 1 µg/ml NI-302.7A8 on a 21-spot membrane, (K) 1 µg/ml NI-302.78H12 on a 16-spot membrane, (L) 1 µg/ml NI-302.71F6 on membrane with 16 spots, (M) 1 µg/ml NI-3O2.11H6 on membrane with 21 spots, (N) 1 µg/ml NI-302.18A1 on membrane with 21 spots, (O) 1 µg/ml NI-302.3 D8 on 21-spot membrane, (P) 1 µg/ml NI-302.46C9 on 21-spot membrane and (Q) 1 µg/ml NI-302.52C9 on 21-spot membrane, (R) 1 µg/ml NI- 302.2A2 on a 21-spot membrane, (S) 1 μg/mL NI-3O2.15E8 on a 21-spot membrane, and (T) 1 μg/mL NI-302.15D3 on a 21-spot membrane.

Fig. 24: Immunohistochemical analysis of NI-302 antibodies shows pronounced staining of intranuclear inclusions of neurons in striatal neurons at a late stage of the disease in Tg(HDexon1)62Gpb/1J transgenic animals using a concentration of 5 nM (74C11, 39C12, 11A4, 22H9, 78H12, 37C12, 7D8, 72F10) or 50 nM (15F9, 71F6, 55D8, 44D7, 7A8, 64E5). Mab5492 is a commercially available antibody against the N-terminus of HTT.

Fig. 25: Determination of the main characteristics of the R6/1 transgenic Tg(HDexon1)61Gpb/J mouse model. (A) Survival curve, (B) body weight curve, and (C) total brain tissue wet weight during disease progression in the indicated animal model. (D-H) Determination of the presence of intranuclear inclusions of neurons during disease progression in the striatum by staining with the anti-HTT antibody NI-302.33C11.

Fig. 26: Determination of the main characteristics of the B6C3Tg(HD82Gln)81Dbo/J (N171-82Q) transgenic mouse model. (A) Survival curve, (B) body weight curve during disease progression, and (C) total wet weight of brain tissue at the end stage of the indicated animal model. (D-F) Determination of the presence of neuronal intranuclear inclusions during disease progression in the striatum by staining with the anti-HTT antibody Mab5492.

Fig. 27: Immunohistochemical analysis at 50 nM NI-302.33C11 (a polyproline epitope) shows intranuclear neuronal inclusion staining in cortical neurons from four different Huntington's disease patients (A-D) and in striatal neurons from B6.Cg-Tg(HDexon1) transgenic animals )61Gpb/J) at a late stage of the disease, 270 days old, at a concentration of 1 (E) and 5 nM (F). No staining is detected in non-transgenic littermates (G) unless the primary antibody (H) is used or control non-Huntington's disease tissue is stained with NI-302.33C11 at 50 nM.

Fig. 28: Immunohistochemical analysis at 50 nM NI-302.63F3 (proline-rich domain epitope) shows staining of neuronal intranuclear inclusions (A-C) and staining of some neurites (D) of cortical neurons in four different Huntington's disease patients (A-D) and in striatal neurons of transgenic animals B6.Cg-Tg(HDexon1)61Gpb/J) at the late stage of the disease, 270 days old, at a concentration of 1 nM (E) and 50 nM (F). No staining is detected in non-transgenic littermates (G) unless the primary antibody (H) is used or control non-Huntington's disease tissue is stained with 50 nM NI-302.63F3.

Fig. 29: Immunohistochemical analysis with 100 nM NI-302.35C1 (terminal epitope of exon 1) shows staining of intranuclear inclusion neurons (A-C) and staining of some neurites (D) of cortical neurons in four different patients with Huntington's disease (A-D) and in striatal neurons transgenic animals B6.Cg-Tg(HDexon1)61Gpb/J) at a late stage of the disease, 270 days old at a concentration of 1 nM (E) and 50 nM (F). No staining is detected in non-transgenic littermates (G) unless the primary antibody (H) is used or control non-Huntington's disease tissue is stained with 100 nM NI-302.35C1.

Fig. 30: Immunohistochemical analysis with commercially available poly-Q antibody Mab1574 (1:2000, Chemicon) shows staining of neuronal intranuclear and cytoplasmic incl.

- 9 046697 tions and staining of some neurites (A, D) of cortical neurons in four different patients with Huntington's disease (A-D) and in striatal neurons of presymptomatic transgenic animals B6.Cg-Tg(HDexon1)61Gpb/J) at a late stage of the disease aged 150 days (E) and 270 days (F). No staining is detected in nontransgenic littermates (G) unless the primary antibody (H) is used or control tissue samples without Huntington's disease are stained with Mab 1574.

Fig. 31: Determination of EC50 of human-derived HTT antibodies for aggregated HD49 (·), aggregated HD21 (), soluble GST-HD49 (A) and GST-HD21 (▼) exon 1-encoded Htt proteins using direct ELISA. Some antibodies (for example, NI-302.64E5 (A) or NI302.7D8 (B)) appear to bind primarily to the non-truncated GST-HD49 protein, indicating that these antibodies preferentially recognize constructs with non-truncated soluble GST-BG containing longer polyglutamine repeats. Antibody NI-302.72F10 (C) shows preferential binding to HD21 constructs; Some antibodies (e.g., NI302.4A6 (D), NI-3O2.12H2 (E), or NI-302.8M1 (E)) exhibited high binding affinities, with similar EC values, to all HTT formulations, suggesting that they bind to an epitope similar to that exposed on aggregated non-truncated HTT exon 1-encoded constructs in an ELISA.

Fig. 32: Characterization of antibodies (A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (D) NI-302.4A6, (E) NI-30212H2, (F) NI-302.8 M1 and (G) NI-302.33C11 (as control) for in vitro time-resolved aggregation reaction products of HD21, HD35 and HD49 using dot blot (left) and filter delay analysis (right) with preferential binding in specifically, NI-302.64E5 and NI-302.72F10 with late (aggregated) reaction products HD35 and/or HD49 in dot blot assays and with SDS-stable aggregates HD35 and HD49 in filter delay assays.

Fig. 33: Target specificity assay using direct ELISA. Antibodies NI-302 (A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (E) NI-302.12H2 and (F) NI-302.8M1 do not bind irrelevant aggregating protein targets, as shown in a direct ELISA binding specificity assay, with the exception of (D) NI-302.4A6 showing some level of p53 binding.

Fig. 34: Study of the effects of the C-terminal binding antibody NI 302.35C1 on behavioral performance during task-directed learning and sensorimotor abilities in a mouse model of HD. (A) The elevated plus maze test was used to examine anxiety levels in R6/1 mice. At 6 months of age, NI302.35C1-treated R6/1 mice spent less time in the open arms of the maze, entered the open arms less frequently, and made fewer unprotected head tilts in the open arm compared to foundation-treated R6/1 animals. Thus, NI-302.35d-treated R6/1 mice exhibited a greater anxiety phenotype compared to non-transgenic littermates. (B) NI-302.35C1-treated R6/1 animals exhibited improved performance in the vertical bar test compared to vehicle-treated R6/1 animals, reaching levels similar to those of nontransgenic animals.

Fig. 35: Determination of the binding epitope of the NI-302 antibody by scanning overlapping peptides. Top: Pepscan image after hybridization of antibody NI-302. Bottom: General images of peptide sequences are shown. The overlapping amino acids in different peptides (putative binding epitope) recognized by antibody NI-302 are given in the consensus sequence below. The HRP-conjugated donkey anti-human IgG Fcy detector antibody does not by itself bind any of the linear huntingtin peptides. (A) NI-302.64E5, (B) NI-302.7D8, (C) NI-302.72F10, (D) NI-302.4A6, (E) NI-3O2.12H2, (F) NI-302.8M1, all antibodies at a concentration of 1 μg/ml, on a membrane with 21 spots.

Fig. 36: Amino acid sequence alignment of CDR regions in the VH and V _L or V _K chains of NI-302 antibodies. Each sequence was checked for the presence of conserved amino acids, segments, or other motifs indicating accumulation of tyrosine residues in the CDR regions.

Detailed Description of the Invention

The present invention relates generally to immunotherapy and non-invasive methods for detecting diseases and/or disorders, as well as conditions associated with the presence of pathological, often mutant and/or aggregated forms of huntingtin (HTT). More specifically, the present invention relates to recombinant human-derived monoclonal antibodies and HTT-binding fragments thereof, synthetic and biotechnological derivatives, which have been derived from sequence information obtained from selected human donor populations and are capable of binding to such isoforms of HTT and its antigens. The recombinant human-derived monoclonal antibody of the present invention provides the advantage of specifically binding to mutated and/or aggregated HTT species and/or fragments thereof, allowing for targeting of pathologically altered HTT species for treatment or diagnosis. There is reason to believe that, due to its human origin, the recombinant antibodies produced according to the present invention can be effective and safe when used as a therapeutic agent, and highly specific as a diagnostic reagent for the detection of pathological NTT without false-positive results.

In addition, the present invention relates to the use of a human monoclonal antibody and any derivatives thereof described herein in the treatment of patients, alone or in combination with other agents used for symptoms associated with NTT amyloidosis, characterized in that the antibody of the present of the invention and any derivatives thereof are designed for simultaneous administration with the side effect suppressing agent, or sequential administration before or after the administration of the specified antibody or derivatives thereof. In this context, the anti-HTT antibody and HTT-binding fragment of the present invention are preferably substantially non-immunogenic in humans. According to one embodiment of the present invention, pharmaceutical compositions are provided containing both a human monoclonal antibody of the present invention or any derivatives thereof and one or more drugs used for symptoms associated with NTT amyloidosis.

I. Definitions

Unless otherwise noted, the definitions of terms in this document are as defined in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0198506732 .

It should be noted that terms given in the singular mean one or more; for example, the term antibody implies one antibody or more antibodies. Thus, the terms singular, the term one or more, and at least one may be used interchangeably herein.

Huntingtin (HTT), also known as IT15, is a disease gene associated with Huntington's disease (HD), a neurodegenerative disorder characterized by the loss of striatal neurons. HD is thought to be caused by an extended, unstable trinucleotide repeat in the HTT gene, which is translated into a polyglutamine repeat in the protein product. A fairly wide range of trinucleotide repeat numbers was determined for normal controls; repeat numbers greater than 35–40 have been described as pathological. The HTT locus (NG_009378.1; 4830-174286; NCBI RefSeqGene) is significant in size, spanning 180 Kb and consisting of 67 exons.

In this context, it has been demonstrated that the N-terminal fragment of the mutant HTT, i.e. the protein encoded by exon 1 of the HTT gene, extended by CAG repeats, is a toxic species of HTT sufficient to promote aggregation and a progressive neurological phenotype in transgenic mice; see, for example, Mangiarini et al., Cell 87 (1996), 493-506 and Ross et al., Lancet Neurol. 10 (2011), 83-98, DiFiglia et al., Science 277 (1997), 1990-1993, Gutekunst et al., J Neurosci 19(7) (1999), 2522-2534.

Unless otherwise stated, antibodies that specifically recognize HTT, antibody specific for/for HTT, and anti-HTT are defined as antibodies that generally bind specifically to HTT, where HTT refers to different forms of HTT, including including, but not limited to, the native form of NTT, as well as other forms of NTT, for example, pathologically altered NTT, such as mutated, misfolded and/or aggregated NTT. The present invention provides human-derived antibodies that are selective for full-length HTT and/or fragments of HTT, and/or mutated, misfolded and/or aggregated forms of HTT.

Unless specifically stated otherwise, the term HTT is used interchangeably to specifically refer to different forms of huntingtin (HTT). The term HTT is also used to generically refer to other HTT conformers, such as pathologically altered forms of HTT, such as misfolded forms of HTT and/or aggregated forms of HTT. In addition, unless specifically stated otherwise, the term HTT refers specifically to HTT encoded by exon 1, and soluble HTT refers to the corresponding GST fusion proteins. The term NTT is also used to collectively refer to all types and forms of NTT, such as mutated NTT. Symbols added to the beginning of terms, such as the term HTT, are used to indicate the organism from which a particular ortholog is derived, for example, hHTT in the case of human HTT or mHTT in the case of mouse-derived HTT.

Antibodies to HTT as described herein specifically bind HTT and its epitopes, as well as various conformational forms of HTT and their epitopes. For example, antibodies are described herein that specifically bind abnormal HTT species or fragments thereof, such as mutated, misfolded and/or aggregated forms of HTT or fragments thereof. The terms (pathologically) mutated, misfolded, and/or aggregated/aggregates of HTT are used interchangeably to specifically refer to the above forms. The term (pathological) aggregated forms or aggregates as used herein describes accumulation products or the formation of clusters as a result of erroneous/pathological interaction of NTTs with each other. Said aggregates, clots or cluster forms may essentially consist or consist of both HTT and/or HTT fragments and non-fibrillar oligomers and/or fibrillar oligomers and fibrils thereof. As used herein, reference to an antibody that specifically binds, selectively binds, or preferentially binds HTT refers to an antibody that does not bind other irrelevant proteins. In one example, an anti-HTT antibody as described herein is capable of binding HTT or an epitope thereof and does not exhibit binding greater than 2-fold above background for other proteins. In a preferred embodiment, the antibody of the present invention does not substantially recognize irrelevant amyloid-forming proteins selected from the group consisting of paired coiled-coil filament (PSF)-tau, TAU, alpha-synuclein, TAR-DNA binding protein 43 (TDP43), islet amyloid polypeptide (IAPP), transthyretin (TTR), serum amyloid A (SAA); see examples 8, 13, 18 and 31. An antibody that specifically binds or selectively binds an HTT conformer refers to an antibody that does not bind all conformational forms of HTT, i.e. does not bind at least one other HTT conformer. For example, disclosed herein are antibodies capable of preferentially binding to mutated and/or aggregated forms of NTT, both in vitro and in tissues obtained from patients with, or at risk for, diseases and/or disorders associated with NTT amyloidosis. or disorders associated with NTTamyloidosis. According to another embodiment of the present invention, the antibodies of the present invention specifically target different regions of HTT encoded by exon 1; see, for example, Fig. 5, 9, 12, 14, 15. Since the anti-HTT antibodies of the present invention were obtained from human subjects, they may also be referred to as human autoantibodies or human-derived antibodies to emphasize that the antibodies were actually originally expressed by the subjects and not are synthetic constructs obtained, for example, using phage libraries to express human immunoglobulins, or xenogeneic antibodies produced by transgenic animals expressing part of the human immunoglobulin repertoire, which has hitherto been one of the common methods when trying to obtain antibodies similar to human antibodies. Alternatively, a human-derived antibody of the present invention may be referred to as synthetic, recombinant and/or biotechnological to distinguish it from actual human serum antibodies, which may be purified on a Protein A column or affinity chromatography column.

However, a particular advantage of the therapeutic approach of the present invention is that the antibodies of the present invention are derived from B cells or memory B cells of healthy human subjects without evidence of a disease in which misfolded/aggregated HTT occurs, or a disease associated with misfolded/aggregated NTT, and, accordingly, these antibodies are likely to prevent clinical manifestations of a disease associated with misfolded/aggregated NTT, or reduce the risk of clinical disease, or delay the onset or progression of clinical manifestations of the disease. Typically, the antibodies of the present invention have also successfully undergone somatic maturation, i.e. optimization of selectivity and efficiency due to high binding affinity of the target HTT molecule through somatic mutations of the variable regions of the antibody. The fact that in vivo, for example in humans, such cells were not activated by congeners or other physiological proteins or cellular structures to cause an autoimmune or allergic reaction is also of great medical significance, since it indicates a significant increase in the likelihood of successful passage through the clinical trial phases. Efficacy, acceptability and tolerability can be said to have already been demonstrated prior to preclinical and clinical development of a prophylactic or therapeutic antibody in at least one human subject. Therefore, target specific efficacy can be expected when used as a therapeutic agent , along with reducing the likelihood of side effects, significantly increase the clinical likelihood of successful use of human-derived anti-HTT antibodies according to the present invention.

At the same time, antibodies originating from a cDNA library or phage displays are artificial molecules, such as a humanized antibody, which nevertheless originates from a mouse and is therefore foreign to the human body. Accordingly, the clinical utility and effectiveness of therapeutic antibodies may be limited by the production of anti-drug antibodies (ADA), which can affect the effectiveness and pharmacokinetics of these antibodies and sometimes lead to serious side effects, see, for example, Igawa et al., MAbs. 3 (2011), 243-252. In particular, humanized antibodies or antibodies produced using recent human antibody technologies, as opposed to human-derived antibodies such as the antibodies of the present invention, tend to stimulate antibody synthesis; It has been reported that said human antibody-like antibodies obtained with e.g.

- 12 046697 using phage display, such as adalimumab, induce the synthesis of ADA antibodies, see, for example,

Mansour, Br. J. Ophthalmol 91 (2007), 274-276 and Igawa et al., MAbs. 3 (2011), 243-252. Accordingly, human-derived antibodies that do not exhibit a tendency to stimulate an undesirable immune response are more beneficial to the patient than artificial molecules derived from libraries or displays.

The term peptide also includes the terms polypeptide and protein (which may sometimes be used interchangeably herein). Likewise included are protein fragments and polypeptides, which may be referred to herein as peptides. However, the term peptide preferably refers to a polymer of amino acids comprising at least 5 consecutive amino acids, preferably at least 10 consecutive amino acids, more preferably at least 15 consecutive amino acids, even more preferably at least 20 consecutive amino acids, and especially preferably at least 25 consecutive amino acids. In addition, the peptide in accordance with the present invention typically contains no more than 100 consecutive amino acids, preferably less than 80 consecutive amino acids, and more preferably less than 50 consecutive amino acids.

Polypeptides:

As used herein, the term polypeptide covers a single polypeptide as well as a collection of polypeptides, and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of product. Accordingly, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to designate a chain or chains of two or more amino acids are included in the definition of polypeptide; the term polypeptide may be used in place of or interchangeably with any of these terms.

The term polypeptide also refers to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation and derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a given nucleic acid sequence. It can be obtained by any method, including chemical synthesis.

The size of the polypeptide of the present invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more more, or 2000 or more amino acids. Polypeptides may differ in a particular three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded polypeptides, and polypeptides that do not have a defined three-dimensional structure, but can take on a significant number of different conformations, are called unfolded polypeptides. As used herein, the term glycoprotein refers to a protein associated with at least one carbohydrate moiety that is attached to said protein via an oxygen-containing or nitrogen-containing side chain or amino acid residue, such as a serine residue or an asparagine residue.

By isolated polypeptide or a fragment, variant or derivative thereof is meant a polypeptide that is outside its natural environment. No specific level of cleansing is required. For example, the isolated polypeptide may be recovered from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced using recombinant DNA techniques, i.e. obtained from microbial or mammalian cells transformed with expression constructs with exogenous recombinant DNA encoding a fusion protein including the desired peptide. The proteins or peptides expressed in most bacterial cultures generally do not contain glycans. Proteins or polypeptides expressed in yeast may have a glycosylation pattern that is different from that of proteins or polypeptides expressed in mammalian cells.

The polypeptides of the present invention include fragments, derivatives, analogs or variants of the polypeptides described above, as well as any combinations thereof. The terms fragment, variant, derivative and analog include peptides and polypeptides with an amino acid sequence sufficiently similar to the amino acid sequence of the naturally occurring peptide. The term sufficiently similar means that a first amino acid sequence that contains a sufficient or minimal number of identical or equivalent amino acid residues to a second amino acid sequence such that the first and second amino acid sequences share a common structural domain and/or differ in common functional activity. For example, amino acid sequences that contain a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65% identical to at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 99% approximately 100% are defined herein as being reasonably similar. Preferably, the variants are sufficiently similar to the amino acid sequence of the preferred peptides of the present invention, in particular, HTT, variants, derivatives or analogs of any of the above. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides whose amino acid sequence differs from the native and wild-type peptide, respectively, by one or more deletions, additions and/or substitutions of amino acids. They can be represented by naturally occurring variants, as well as artificially constructed variants.

In addition, the terms fragment, variant, derivative and analogue with respect to antibodies or antibody polypeptides of the present invention include any polypeptides that retain at least some of the antigen binding properties of the corresponding native binding molecule, antibody or polypeptide. The polypeptide fragments of the present invention include proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein. Variants of antibodies and antibody polypeptides according to the present invention include fragments as described above, as well as polypeptides with altered sequences due to amino acid substitutions, deletions or insertions. Variants can either occur naturally or be variants that do not occur in nature. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Polypeptide variants may include conservative or non-conservative substitutions, deletions or additions of amino acids. Derivatives of specific HTT-binding molecules, for example, antibodies and antibody polypeptides of the present invention, are polypeptides that have been modified in such a way that they exhibit additional features not found in the native polypeptide. Examples include fusion proteins. Polypeptide variants may also be referred to herein as polypeptide analogs. As used herein, a derivative of a binding molecule or fragment thereof, an antibody, or an antibody polypeptide refers to a subject polypeptide containing one or more residues chemically derivatized by reaction with a functional side group. Also included as derivatives are peptides that contain one or more naturally occurring derivatives of the twenty standard amino acids. For example, proline can be replaced by 4-hydroxyproline; 5-hydroxylysine can be replaced by lysine; histidine can be replaced by 3-methylhistidine; serine can be replaced by homoserine; and lysine can be replaced by ornithine.

Determination of similarity and/or identity of molecules:

The similarity of two peptides is determined by comparing the amino acid sequence of one peptide with the sequence of a second peptide; see Example 35 and also FIG. 36. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical to the specified amino acid or is represented by a conservative amino acid substitution. Conservative substitutions include those described in: Dayhoff, M.O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups correspond to conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu.

The similarity of two polynucleotides is determined by comparing the nucleic acid sequence of one polynucleotide with the sequence of the other polynucleotide. The nucleic acid of one polynucleotide is similar to the corresponding nucleic acid of a second polynucleotide if it is identical or, if the nucleic acid is part of a coding sequence, the corresponding triplet in the said nucleic acid encodes the same amino acid or a conservative amino acid substitution.

Determination of the percentage identity or similarity of two sequences is preferably carried out using the Karlin-Altschul mathematical algorithm (Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877). This algorithm is included in the BLASTn and BLASTp programs (Altschul et al. (1990) J. Mol. Biol. 215: 403-410) provided by the National Center for Biotechnology Information (NCBI; https://blast.ncbi.nlm .nih.gov/Blast.cgi).

Determination of percent identity or similarity is performed using standard parameters of the BLASTn polynucleotide search programs and the BLASTp protein BLAST search programs, as recommended on the NCBI web page and in the BLAST Program Selection Guide for sequences of a certain length and composition.

BLAST polynucleotide searches are performed using the BLASTn program.

General parameters: the value in the max target sequences window can be defined as 100, the short queries parameter can be enabled, the value in the expect threshold window can be defined as 1000; and the value in the word size window can be defined as 7, as recommended for short sequences (less than 20 bases) on the NCBI web page. For longer sequences, the value in the wait threshold window can be defined as 10, and the value in the segment length window can be defined as 11. Score parameters: Match/mismatch Scores values can be defined as 1, -2; The Gap Costs can be defined as linear. Filters and Masking: Low complexity regions option can be disabled; the Species-specific repeats option can be disabled; the Mask for lookup table only parameter can be enabled, the DUST Filter Settings parameter can be enabled; and the Mask lower case letters option can be disabled. In the general case, Search for short nearly exact matches can be used, which includes most of the described settings. Refer to the BLAST Program Selection Guide published on the NCBI webpage for additional relevant information.

BLAST protein searches are performed using the BLASTp program. General parameters: the value in the max target sequences window can be defined as 100, the short queries parameter can be enabled, the value in the expect threshold window can be defined as 10; and the value in the segment length window can be defined as 3. Evaluation parameters: in the Matrix window, the BLOSUM62 option can be selected, the values in the gap penalties window are defined as 11 for introducing a gap and 1 for continuing a gap, Compositional adjustments ) can be defined as Conditional compositional score matrix adjustment. Filters and Masking: Low complexity regions option can be disabled, Mask for lookup table only option can be disabled; and the Mask lower case letters option can be disabled.

Modifications to both programs, such as those regarding the length of the search sequences, are made in accordance with the recommendations in the BLAST Program Selection Guide, available in HTML and PDF formats on the NCBI web page.

Polynucleotides:

The term polynucleotide includes a single nucleic acid as well as a collection of nucleic acids, and refers to an isolated nucleic acid molecule or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The polynucleotide may include a standard phosphodiester linkage or a non-standard linkage (eg, an amide linkage, such as found in peptide nucleic acids (PNA)). The term nucleic acid refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. An isolated nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been extracted from its natural environment. For example, a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated for purposes of the present invention. Additional examples of isolated polynucleotide include recombinant polynucleotides contained in heterologous host cells, or purified (partially or substantially) polynucleotides in solution. The isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids in accordance with the present invention further include such molecules obtained synthetically. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element such as a promoter, a ribosome binding site, or a transcription terminator.

As used herein, a coding region means a portion of a nucleic acid that consists of codons translated into amino acids. Although a stop codon (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc. are not included in the coding region. Two or more coding regions of the present invention may be located on a single polynucleotide construct, eg on a single vector, or on separate polynucleotide constructs, eg on separate vectors. In addition, any vector may contain one coding region, or may contain two or more coding regions, for example, one vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid of the present invention may encode heterologous coding regions, whether or not connected to a nucleic acid encoding a binding molecule, antibody, or fragment, variant, or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

In certain embodiments, said polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide containing a nucleic acid that encodes a polypeptide may typically include a promoter and/or other transcriptional or translational control elements operably linked to one or more coding regions. Functional linkage means that the coding region of a gene product, such as a polypeptide, is linked to one or more regulatory sequences such that expression of the gene product is influenced or controlled by said regulatory sequence(s). Two DNA fragments (e.g., the coding region of a polypeptide and its associated promoter) are operably linked if induction of promoter function results in the transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not impair the ability of expression regulatory sequences to direct expression of the gene product or does not affect the possibility of transcription of the DNA template. Accordingly, a promoter region is operably linked to a nucleic acid encoding a polypeptide if said promoter is capable of causing transcription of said nucleic acid. The promoter may be a cell-specific promoter that directs DNA transcription substantially only in certain cells. Other transcriptional control elements in addition to promoters, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to a polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are described herein.

Various areas of transcription control are known to those skilled in the art. These regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, the promoter and enhancer segments of cytomegaloviruses (immediate early promoter, in combination with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, promoters inducible by interferons or interleukins).

Likewise, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, transcription initiation and termination codons, and picornavirus-derived elements (particularly the internal ribosome entry site, or IRES, also called the CITE sequence).

In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).

The polynucleotides and nucleic acid coding regions of the present invention may be linked to additional coding regions encoding secretory or signal peptides that direct secretion of the polypeptide encoded by the polynucleotide of the present invention. According to the signal sequence hypothesis, proteins secreted by mammalian cells contain a signal peptide or secretory leader sequence that is cleaved from the mature protein after the nascent protein chain has begun to be exported through the rough endoplasmic reticulum. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells typically contain a signal peptide linked to the N-terminus of the polypeptide, which is cleaved from the complete or full-length polypeptide to produce the secreted or mature form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative thereof that retains the ability to direct secretion of a operably related polypeptide, is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by a human tissue plasminogen activator (TPA) or mouse β-glucuronidase leader sequence.

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A binding molecule in the context of the present invention refers generally to antibodies and fragments thereof, but may also refer to other non-antibody molecules that bind to HTT, including, but not limited to, hormone molecules, receptors, ligands, ankyrins, the major histocompatibility complex (MHC), chaperones such as heat shock proteins (HSPs), and intercellular adhesion molecules such as members of the cadherin, integrin, C-type lectin, and immunoglobulin (Ig) superfamilies. Accordingly, for purposes of illustration only and without limiting the scope of the present invention, most of the embodiments presented below describe antibodies and antibody-like molecules that are preferred binding molecules in the development of therapeutic and diagnostic agents.

Antibodies:

The terms antibody and immunoglobulin are used interchangeably herein. An antibody or immunoglobulin is a binding molecule that contains at least a heavy chain variable domain, and typically contains at least a heavy chain and a light chain variable domain. The basic immunoglobulin structures of vertebrates are relatively well studied; see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As described in more detail below, the term immunoglobulin includes various broad classes of polypeptides that may differ biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ,μ, α, δ, ε), and include a number of subclasses (eg, γ1-γ4). The nature of this chain determines the class of the antibody: IgG, IgM, IgA IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), for example, lgG1, lgG2, lgG3, lgG4, lgA1, etc., are described in detail and are known to provide functional specialization. Modified versions of each of these classes and isotypes will be readily apparent to those skilled in the art in light of the present disclosure and are accordingly intended to be included within the scope of the present invention. All classes of immunoglobulins are expressly included within the scope of the present invention; the following description applies mainly to immunoglobulin molecules of the IgG class. In the case of IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides having a molecular weight of approximately 23,000 Da and two identical heavy chain polypeptides having a molecular weight of 53,000-70,000. These four chains are typically linked by disulfide bonds to form a Y''-shaped configuration in which the light chains flank the heavy chains, starting at the Y extension and continuing along the variable region.

Light chains are classified as kappa or lambda (κ,λ). Any class of heavy chain can bind to either a kappa light chain or a lambda light chain. In general, the light and heavy chains are covalently linked, and the tail portions of the two heavy chains are linked to each other through covalent disulfide bonds or non-covalent bonds when the immunoglobulins are produced using hybridomas, B cells or genetically engineered host cells. The heavy chain amino acid sequences begin at the N-terminus in the Y-shaped fork region and proceed towards the C-terminus located at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms constant and variable are used in a functional sense. In this regard, it should be understood that both the light (VL) and heavy (VH) chain variable domains determine antigen recognition and specificity. In turn, the light chain (CL) and heavy chain constant domains (CH1, CH2 or CH3) provide important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, constant region domains are numbered in the direction away from the antigen-binding site or amino terminus of the antibody. The N-terminal part is the variable region; The terminal portion is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains, respectively. As noted above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. Accordingly, the V _L domain and the V H domain, or set of complementarity determining regions (CDRs) of an antibody, in combination form a variable region defining a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y-shaped antibody. More specifically, the antigen binding site is defined by three CDR regions on each of the VH and VL chains. Any antibody or immunoglobulin fragment that contains a structure sufficient to specifically bind to an HTT is interchangeably referred to herein as a binding fragment or an immunospecific fragment.

Naturally occurring antibodies contain six hypervariable regions, sometimes called complementarity determining regions, or CDRs, present in each

- 17 046697 house of the antigen-binding domain and are short sequences of amino acids containing non-contiguous sections, located in a specific manner, ensuring the formation of an antigen-binding domain when the antibody takes on a three-dimensional configuration in an aqueous environment. The CDR regions are flanked by four relatively conserved framework regions, or FRs, that exhibit less intermolecular variability. The framework regions generally have a β-sheet conformation, and the CDR regions form loops connecting and, in some cases, forming part of the specified β-sheet structure. Accordingly, the function of the framework regions is to form a scaffold that ensures that the CDR regions are positioned in the correct orientation through interchain non-covalent interactions. The antigen-binding domain, formed by the arranged CDRs, determines the surface complementarity to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to the cognate epitope. The amino acids constituting the CDR and framework regions, respectively, of any heavy or light chain variable region can be readily identified by one skilled in the art since they have been precisely defined; see sources: Sequences of Proteins of Immunological Interest, Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference in their entirety.

In the event that there are two or more definitions of a term that is used and/or accepted in the art, the definition of the term herein includes all such meanings unless expressly stated otherwise. A specific example is the use of the term complementarity determining region (CDR) to describe non-contiguous antigen binding sites found in both heavy and light chain variable region polypeptides. This particular area has been described in: Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983), and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, incorporated herein by reference, the definitions including overlapping residues or sets of residues of the amino acid sequences being compared. It is intended that the use of any term relating to the CDR of an antibody or variants thereof is within the scope of the term as defined and used herein. Suitable amino acid residues constituting the CDR regions, as defined in each of the above references, are listed below in table. I for comparison. The exact number of residues that make up a particular CDR region varies depending on the sequence and size of the CDR. Those skilled in the art can, using standard techniques, determine the residues constituting a particular hypervariable region or CDR of a human IgG subtype antibody based on the amino acid sequence of the variable region of said antibody.

Table I. CDR ¹ Definitions

Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

¹ Numbering of all CDR areas in table. I follows the numbering conventions of Kabat et al. (see below).

In Kabat et al. A variable domain sequence numbering system applicable to any antibody is also described. One skilled in the art can unambiguously determine any variable domain sequence using the specified Kabat numbering system without recourse to any experimental data other than the sequence itself. Kabat numbering in this document refers to the numbering system presented in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983). Unless otherwise indicated, the numbering given for specific amino acid positions of an antibody or antigen binding fragment, variant or derivative thereof according to the present invention follows the Kabat numbering system, which, however, is theoretical and may not be equally applicable to every antibody. For example, depending on the position of the first CDR region, subsequent CDR regions may be shifted in any direction.

In addition to a human-derived monoclonal antibody or an antigen-binding fragment thereof, a synthetic or biotechnological derivative of said antibody, which are provided in particular by preferred embodiments of the present invention, antibodies

- 18 046697 or antigen binding fragments, immunospecific fragments, variants or derivatives according to the present invention include, but are not limited to, polyclonal, monoclonal, multispecific antibodies, human antibodies, humanized, primate antibody elements, mouse antibody elements or hybrid antibodies, single chain antibodies , epitope-binding fragments, e.g. Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain disulfide-linked Fv (sdFv), fragments containing a VL or VH domain, fragments derived from from a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-idiotypic antibodies to antibodies as described herein). ScFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019. The immunoglobulin or antibody molecules of the present invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or a subclass of immunoglobulin molecules.

In one embodiment, the antibody of the present invention is not IgM or a derivative thereof with a pentavalent structure. More specifically, according to specific embodiments of the present invention, particularly in therapeutic applications, the use of IgM is less advisable than the use of IgG and other bivalent antibodies or corresponding binding molecules, since IgM, due to its pentavalent structure and lack of affinity maturation, often exhibits nonspecific cross-linking. reactivity and very low affinity.

In a particular preferred embodiment, the antibody of the present invention is not a polyclonal antibody, i.e. essentially formed by one specific type of antibody, rather than a mixture obtained from a sample of plasma immunoglobulins.

Antibody fragments, including single chain antibodies, may contain variable region(s), alone or in combination with the following complete or partial sequences: hinge region, CH1, CH2 and CH3 domains. Also included in the present invention are HTT binding fragments that contain any combination of variable region(s) with a hinge region, CH1, CH2 and CH3 domains. The antibodies or immunospecific fragments thereof of the present invention can be derived from any animal, including birds and mammals. Preferably, said antibodies are derived from human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another embodiment, said variable region may be derived from cartilaginous fish (eg, sharks).

In one aspect, the antibody of the present invention is a human-derived monoclonal antibody obtained from a human, wherein a B cell expressing said antibody, and in turn, an antibody or preferably a cDNA encoding a variable domain and, optionally, a cDNA of a cognate constant domain , received from a person. Optionally, the human antibody framework region is aligned and mapped to suitable human germline variable region sequences from the database; see, for example, the Vbase (https://vbase.mrc-cpe.cam.ac.uk/) (https://www.vbase2.org/) of the MRC Center for Protein Engineering, Cambridge, UK). For example, amino acids potentially derived from the true germline sequence may actually be derived from PCR primer sequences included during the cloning process. Compared with artificially produced antibodies similar to human antibodies, for example, single chain antibody fragments (scFv) obtained from a phage display antibody library or using xenogeneic mice, the human monoclonal antibody of the present invention is characterized by (i) production using the immune response of human, and not surrogate animals, i.e. the antibody was synthesized in response to a naturally occurring NTT in a relevant conformation in humans, (ii) protects the individual or at least has a significant effect on the NTT present, and (iii) since the antibody is of human origin, there are risks of cross-reactivity with its own antigens are minimized. Accordingly, in accordance with the present invention, the terms human monoclonal antibody, human monoclonal autoantibody, human antibody and the like are used. is used to refer to an HTT-binding molecule that is human-derived, i.e. either was isolated from a human cell, such as a B cell or a B cell-based hybridoma, or its cDNA was cloned directly from the mRNA of a human cell, such as a human memory B cell. A human antibody remains human, i.e. originating from humans, even if amino acid substitutions are made, for example to improve binding characteristics. In this context, unlike humanized antibodies and other human-like antibodies, see also the discussion above, the human-derived antibodies of the present invention are characterized by the presence of CDR regions that have been in contact with the human body and, accordingly, there is essentially no risk of immunogenicity. Accordingly, an antibody of the present invention may still be considered to be of human origin if at least one, preferably two, and most preferably all three CDR regions of the light and heavy variable regions, or both

- 19 046697 antibody chains are derived from human antibodies, examples of which are provided herein.

In one embodiment, the human-derived antibodies of the present invention contain regions that are heterologous to naturally occurring antibodies, for example, amino acid substitutions in the framework region, a constant region exogenously linked to a variable region, other amino acids at the C or N termini, and the like. .

Antibodies derived from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulins as described below and, for example, in US Pat. No. 5,939,598 to Kucherlapati et al. are called human-like antibodies to distinguish them from true human antibodies according to the present invention.

For example, heavy-light chain pairing in human-like antibodies, such as synthetic and semi-synthetic antibodies, typically isolated using phage display, does not necessarily reflect the original pairing occurring in the original human B cell. Accordingly, Fab and scFv fragments derived from recombinant expression libraries commonly used in the prior art may be considered artificial, with all possible attendant effects in terms of immunogenicity and stability.

At the same time, the present invention provides isolated affinity-matured antibodies obtained from selected human subjects, characterized by therapeutic utility and tolerability in humans. As used herein, the term rodent antibody element-containing antibody or rodent antibody element-containing immunoglobulin refers to an antibody containing one or more CDR regions from a human antibody according to the present invention; and a human framework region that contains amino acid substitutions and/or deletions and/or insertions based on the rodent antibody sequence. Preferably, the rodent sequences are those derived from mice and rats, and antibodies containing such sequences are referred to as mouse-element-containing or rat-element-containing, respectively. The human immunoglobulin, which is the source of the CDR regions, is called the original, or acceptor, and the rodent antibody, which is the source of the changes in the framework regions, is called the donor. The constant regions do not necessarily have to be present, but if present they are typically substantially identical to the constant regions of the rodent antibody, i.e. are at least about 85-90% identical, preferably about 95% or more. Thus, in some embodiments, a full-length human immunoglobulin containing mouse antibody heavy or light chain elements comprises a mouse constant region, human CDR regions, and a substantially human-derived framework region that contains a number of mouse antibody-corresponding amino acid substitutions. Typically, the mouse antibody element-containing antibody is an antibody having a mouse antibody element-containing variable light chain and/or a mouse antibody element-containing variable heavy chain. For example, an antibody containing mouse antibody elements does not include typical fusion antibodies because the entire variable region of the fusion antibody is not derived from a mouse.

A modified antibody that has been introduced with mouse antibody elements through the process of introducing mouse antibody elements binds to the same antigen as the original antibody from which the CDRs are derived and is generally less immunogenic in mice compared to the original antibody. The above description of mouse antibody element-containing antibodies also similarly applies to rodent antibody element-containing antibodies, eg, rat antibody element-containing antibodies, wherein rat sequences are used instead of mouse sequences.

As used herein, the term heavy chain portion includes amino acid sequences derived from the immunoglobulin heavy chain. The heavy chain portion-containing polypeptide contains at least one of a CH1 domain, a hinge domain (eg, an upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the present invention may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain containing a CH1 domain, at least part of a hinge domain and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. According to another embodiment, the polypeptide of the present invention includes a polypeptide chain containing a CH3 domain. In addition, a binding polypeptide for use in the present invention may lack at least a portion of the CH2 domain (eg, the CH2 domain, in whole or in part). As noted above, one skilled in the art will appreciate that these domains (eg, heavy chain portions) may be modified to differ in amino acid sequence from a naturally occurring immunoglobulin molecule.

- 20 046697

In certain antibodies or antigen binding fragments, variants or derivatives thereof as described herein, the heavy chain portions of one polypeptide chain of the multimer are identical to the heavy chain portions of a second polypeptide chain of the multimer. Alternatively, the monomers comprising the heavy chain portion of the present invention are not identical. For example, each monomer may contain a different target binding site to form, for example, a bispecific antibody or diabody.

As used herein, the term bispecific or bifunctional antibody molecule refers to an antibody molecule containing two different epitope/antigen binding sites and, accordingly, having binding specificity for two different target epitopes. The two epitopes may be epitopes of the same antigen or different antigens. In contrast, a bivalent antibody may contain binding sites that have identical antigenic specificity. Methods for preparing a bispecific antibody are known in the art, for example, chemical conjugation of two different monoclonal antibodies according to Example 36, or, for example, chemical conjugation of two antibody fragments, for example two Fab fragments (Brennan et al., Science 229 (1985), 81 -83; Nitta et al., J. Immunol. 19 (1989), 1437-1441; Glennie et al., J. Immunol. 139 (1987), 2367-2375; Immunol., 21 (1991), 2431-2435). Alternatively, bispecific antibodies are produced recombinantly (Gruber et al., J. Immunol. 152 (1994), 5368-5374; Kurucz et al., J. Immunol. 154 (1995), 4576-4582; Mallender and Voss, J Biol. Chem. 269 (1994), 199-206). Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two pairs of chains, each consisting of an immunoglobulin heavy and a light chain, with the two heavy chains having different binding specificities. By randomly selecting heavy and light chains, a mixture of ten antibodies with different structures is potentially obtained, of which only one has the required binding specificity (Milstein and Cuello, Nature 305 (1983), 537-540; Lanzavecchia and Scheidegger, Eur. J. Immunol. 17 (1987), 105-111. An alternative method involves coupling variable domains with the desired binding specificity to a heavy chain constant region comprising at least a portion of the hinge region, the CH2 and CH3 regions, in one embodiment, said CH1 region containing the site required. for light chain binding, is present in at least one of said hybrids. The DNAs encoding said hybrids, and, if necessary, the light chains are inserted into separate expression vectors and then cotransfected into a suitable host organism. The coding sequences of two or all three may be inserted. chains into one expression vector.

In another embodiment, antibodies or antigen-binding fragments, variants or derivatives thereof as described herein consist of a single polypeptide chain, for example, scFv, and must be expressed intracellularly (intrabodies) for potential therapeutic and diagnostic use in vivo.

The heavy chain portions of the binding polypeptide for use in the diagnostic and therapeutic methods described herein may be derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. According to another example, the heavy chain portion may comprise a hinge region derived partly from an IgG1 molecule and partly from an IgG3 molecule. According to another example, the heavy chain portion may comprise a hybrid hinge derived partly from an IgG1 molecule and partly from an IgG4 molecule.

As used herein, the term light chain portion includes amino acid sequences derived from the immunoglobulin light chain. Preferably, the light chain portion contains at least one of V _L or CL domains.

The minimum size of a peptide or polypeptide epitope for an antibody is believed to be approximately 4-5 amino acids. Peptide or polypeptide epitopes preferably contain at least 7, more preferably at least 9, and most preferably at least about 15 to about 30 amino acids. Because the CDR region is capable of recognizing an antigenic peptide or polypeptide in tertiary form, the amino acids constituting the epitope do not necessarily have to be contiguous and, in some cases, do not even have to be located on the same peptide chain. According to the present invention, the peptide or polypeptide epitope recognized by the antibodies of the present invention contains a sequence comprising at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9 , at least 10, at least 15, at least 20, at least 25, or from about 15 to about 30 contiguous or non-contiguous HTT amino acids, particularly in the N-terminal, polyproline region, proline-rich region, or C -terminal region encoded by exon 1.

By the terms specific binding or specific recognition, used interchangeably herein, it is generally meant that a binding molecule, such as an antibody, binds to an epitope via an antigen binding domain, and that said binding

- 21 046697 naming implies some degree of complementarity between the antigen-binding domain and the epitope. As used herein, an antibody is said to specifically bind an epitope if it binds to said epitope via the antigen binding domain more readily than it would bind to a random irrelevant epitope. The term specificity is used herein to refer to the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be considered to have higher specificity for a particular epitope than antibody B; or antibody A may be stated to bind to epitope C with higher specificity than to the related epitope D.

When using the term immunological binding characteristics or a term for other antibody-antigen binding characteristics, all grammatical forms of the term refer to the specificity, affinity, cross-reactivity and other binding characteristics of the antibody.

By preferential binding is meant that a binding molecule, such as an antibody, specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or similar epitope. Accordingly, an antibody that preferentially binds to a given epitope will be more likely to bind to that epitope than to a related epitope, even though such antibody may cross-link with the related epitope.

According to a non-limiting example of a binding molecule, for example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with a dissociation constant (K _D ) that is less than the KD of said antibody for the second epitope. According to another non-limiting example, an antibody may be considered to preferentially bind a first antigen if it binds said first epitope with an affinity that is at least one order of magnitude less than the KD of said antibody for the second epitope. According to another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with an affinity that is at least two orders of magnitude less than the KD of said antibody for the second epitope.

According to another non-limiting example of a binding molecule, for example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with a dissociation rate (k(off)) less than the antibody k(off) for the second epitope. According to another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope. According to another non-limiting example, an antibody may be considered to preferentially bind a first epitope if it binds said first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or an antigen binding fragment, variant, or derivative as described herein, may be stated to bind HTT or a fragment, variant, or specific conformational form thereof with a dissociation rate (k(off)) of 5x10 ^-2 s ^-1 , 10 ^-2 s ^-1 , 5x10 ^-3 s ^-1 or 10 ^-3 s ^-1 or less. More preferably, the antibody of the present invention may be stated to bind HTT or a fragment, variant or specific conformational form thereof with a dissociation rate (k(off)) of 5x10 ^-4 s ^-1 , 10 ^-4 s ^-1 , 5x10 ^{- 5} s ^-1 , or 10 ^-5 s ^-1 5x10 ^-6 s ^-1 , 10 ^-6 s ^-1 , 5x 10 ^-7 s ^-1 or 10 ^-7 s ^-1 or less.

A binding molecule, such as an antibody or an antigen binding fragment, variant or derivative as described herein, may be stated to bind HTT or fragments, variants or a specific conformational form thereof with an association rate (k(on)) of 10 ³ M ^{- 1} s ^-1 , 5 x10 ³ M ^-1 s ^-1 , 10 ⁴ M ^-1 s ^-1 or 5x10 ⁴ M ^-1 s ^-1 or more. More preferably, the antibody of the present invention may be stated to bind HTT or a fragment, variant or specific conformational form thereof with an association rate (k(on)) of 10 ⁵ M ^-1 s ^-1 , 5 x 10 ⁵ M ^-1 s ^-1 , 10 ⁶ M ^-1 s ^-1 , or 5x10 ⁶ M ^-1 s ^-1 or 10 ⁷ M ^-1 s ^-1 or more.

A binding molecule, such as an antibody, is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to said epitope to such an extent that it blocks, to a certain level, the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, using a competitive ELISA. The antibody may be stated to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term affinity refers to the magnitude of the binding strength of an individual epitope to the CDR region of a binding molecule, e.g., an immunoglobule molecule

- 22 046697 on; see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988), pp. 27-28. As used herein, the term avidity refers to the overall stability of a population of immunoglobulins and an antigen complex, that is, the strength of functional binding of a mixture of immunoglobulins to an antigen; see, for example, Harlow, pp. 29-34. Avidity is related both to the affinity of individual immunoglobulin molecules in a population for specific epitopes and to the valences of immunoglobulins and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen containing an epitope with a highly repetitive structure, such as a polymer, is highly avid. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antibody-Antigen Interactions, Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, NY (1992 ), and the methods described herein. Common techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions, for example, at different salt concentrations, pH values. Accordingly, measurements of affinity and other measures of antigen binding, eg, _KD , _IC50 , are preferably carried out in standardized solutions of antibody and antigen, and using a standardized buffer.

Binding molecules, eg antibodies or antigen binding fragments, variants or derivatives thereof of the present invention may also be described or defined in terms of their cross-reactivity. As used herein, the term cross-reactivity refers to the ability of an antibody specific for one antigen to react with a second antigen; it is a measure of the relatedness of two different antigenic substances. Accordingly, an antibody is cross-reactive if it binds to an epitope different from the one that induced its formation. A cross-reactive epitope typically contains a significant number of complementary structural features identical to the inducing epitope, and in some cases may actually be a better fit than the original one.

For example, certain antibodies may exhibit some degree of cross-reactivity, that is, they bind related but non-identical epitopes, e.g. epitopes that are at least 95% identical to a reference epitope, at least 90% identical, at least 85% identical, at least by at least 80%, by at least 75%, by at least 70%, by at least 65%, by at least 60%, by at least 55% and by at least 50% (as assessed by using methods known in the art and described herein). An antibody may be stated to have little or no cross-reactivity if it does not bind epitopes that are less than 95% identical to the reference epitope, less than 90% identical, less than 85% identical, less than 80% identical, less than 75%, less than 70%, less than 65%, less than 60%, less than 55% and less than 50% (as assessed using methods known in the art and described herein ). An antibody may be considered highly specific for a particular epitope if it does not bind any other analog, orthologue, or homologue of that epitope.

Binding molecules, such as antibodies or antigen binding fragments, variants or derivatives thereof, of the present invention may also be described or defined in terms of their affinity for binding HTT and/or mutated, misfolded and/or aggregated HTT species, and/or their fragments. Preferred binding affinities include those corresponding to a dissociation constant, or Kd, of less than 5 x ^{10 -2} M, 10 ^-2 M, 5 x 10 ^-3 M, 10 ^-3 M, 5 x 10 ^-4 M, 10 ^-4 M, 5 x 10 ^-5 M, 10 ^-5 M, 5x10 ^-6 M, 10 ^-6 M, 5x10 ^-7 M, 10 ^-7 M, 5x10 ^-8 M, 10 ^-8 M, 5x10 ^-9 M, 10 ⁹ M, 5x10 ^-10 M, 10 ^-10 M, 5x10 ^-11 M, 10 ^-11 M, 5x10 ^-12 M, 10 ^-12 M, 5x10 ^-13 M, 10 ^-13 M, 5x10 ^-14 M, 10 ^-14 M, 5x10 ^-15 M or 10 ^-15 M.

As stated above, the subunit structures and three-dimensional configuration of the constant regions of various classes of immunoglobulins are well known. As used herein, the term VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term CIS' domain includes the first (closest to the amino terminus) constant region of the immunoglobulin heavy chain domain. The CH1 domain is adjacent to the VH domain and is located towards the amino terminus relative to the hinge region of the immunoglobulin heavy chain molecule.

As used herein, the term CH2 domain includes the portion of the heavy chain molecule encompassing, for example, antibody residues approximately 244 to 360, according to standard numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat EA et al., cit.). The CH2 domain is unique in that it is not tightly coupled to another domain; CH2 are two N-linked branched carbohydrate chains that are located between the two CH2 domains of the intact native IgG molecule. It is also well known that the CH3 domain extends from the CH2 domain towards the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term hinge region includes the portion of the heavy chain molecule connecting the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be divided into three distinct domains: the upper, middle, and lower hinge domains; see Roux et al., J. Immunol. 161 (1998), 4083-4090.

As used herein, the term disulfide bond includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group capable of forming a disulfide bond, or bridge, with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are joined by a disulfide bond, and the two heavy chains are joined by two disulfide bonds at positions corresponding to positions 239 and 242 according to the Kabat numbering system (positions 226 or 229 in the EU numbering system).

In this document, the terms connected, hybrid, or connection/fusion are used interchangeably. These terms refer to the joining of two or more elements or components by any means, including the use of chemical conjugation or recombinant methods. In-frame fusion refers to the joining of two or more open reading frames (ORFs) of polynucleotides to form a contiguous longer ORF in a manner that ensures that the correct translational reading frame of the original ORF is retained. Accordingly, a recombinant fusion protein is a single protein containing two or more segments that correspond to the polypeptides encoded by the original ORFs (which segments are not typically so linked in nature). Although the resulting reading frame for all combined segments is accordingly contiguous, said segments may be physically or spatially separated, for example by a linker sequence within the reading frame. For example, polynucleotides encoding immunoglobulin variable region CDR regions may be fused in frame but separated by a polynucleotide encoding at least one framework region or additional immunoglobulin CDR regions, provided that the fusion CDRs are translated together as part of a single polypeptide.

The term expression as used herein refers to the process of producing a biochemical substance, such as RNA or a polypeptide, from a gene. This process includes any manifestation of the functional presence of a gene in a cell, including, without limitation, gene knockdown, as well as both transient and stable expression. It includes, but is not limited to, transcription of a gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and translation of mRNA into polypeptide(s). In the event that the final desired product is a biochemical agent, expression involves the production of said biochemical agent and any precursors thereof. Expression of a gene results in the formation of a gene product. As used herein, the gene product may be either a nucleic acid, such as messenger RNA, synthesized by transcription of a gene, or a polypeptide that is translated from the transcript. The gene products described herein further include nucleic acids that have undergone post-transcriptional modifications, such as polyadenylation, or polypeptides that have undergone post-translational modifications, such as methylation, glycosylation, addition of lipids, binding to other protein subunits, proteolytic cleavage, and the like.

As used herein, the term sample refers to any biological material obtained from a subject or patient. In one aspect, the sample may include blood, peritoneal fluid, CSF, saliva, or urine. In other aspects, the sample may include whole blood, blood plasma, blood serum, B cells isolated from blood samples, and cultured cells (eg, B cells obtained from a subject). The sample may also include a biopsy or tissue sample, including nerve tissue. In other aspects, the sample may include whole cells and/or cell lysate. Blood samples can be collected using methods known in the art. In one aspect, the pellet can be resuspended by vortexing at 4°C in a 200 μl volume of buffer (20 mM Tris, pH 7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, protease inhibitor IX Sigma and IX Sigma phosphatase inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes with occasional vortex mixing. After stirring at 15,000 x g for 5 minutes at approximately 4°C, aliquots of the supernatant can be stored at approximately -70°C.

Diseases:

Unless otherwise specified, the terms disorder and disease are used interchangeably herein and include any undesirable physiological change in a subject, animal, isolated organ, tissue, or cell/cell culture.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by extension of the trinucleotide CAG repeat of the huntingtin gene (HTT) (Huntington's Disease Collaborative Research Group, Cell 72(6) (1993) , 971-983), while the threshold for pathogenicity of this extension is approximately 37 repeats, while a smaller number of repeats does not lead to pathogenesis, see for example Trottier et al., Nature 378(6555) (1995), 403- 406. Elongation due to trinucleotides

CAG repeats result in the formation of an extended polyglutamine (poly-Gln, poly-Q) tract at the amino terminus of the huntingtin protein (HTT), which is associated with HTT aggregation. However, the exact mechanism leading to the accumulation of NTT and the appearance of associated symptoms has not yet been established.

Studies have shown that both flanking regions of the polyglutamine (poly-Q) tract, i.e. The amino-terminal region, consisting of an amphipathic alpha-helical targeting domain, and the carboxy-terminal region, characterized by two proline tracts (polyproline region) and a leucine/proline-rich tract (proline-rich region), appear to be critical in mediating the toxicity of the mutated HTT, see for example, Caron et al., PNAS 110 (2013), 1461014615.

Mechanism that contributes to the pathological symptoms of HD, such as hyperkinesia, hypokinesia, mental and movement disorders, including affective and desire disorders, decreased motor stability, irresponsible and impulsive behavior, avoidance of impressions and depression, visual processing disorders, subcortical dementia, decreased cognitive abilities, disorientation and speech disorders, delusional disorders, motor restlessness in the arms, legs, face, head and torso, choreic hyperkinesis, dysarthria, dysphagia, anarthria, dystonia, not yet clear. Possible mechanisms include, but are not limited to, reduced flexibility of the hinge region due to the elongation of the HTT polyglutamine tract, as well as the action of proteases leading to the formation of other HTT fragments as a result of the presence of an elongated polyglutamine tract.

Since the antibodies of the present invention have been shown to be therapeutically effective in a mouse model of HD, see, for example, Example 24 and FIG. 17, as well as example 34 and fig. 34, and, in addition, are able to bind to HTT amyloids in tissue sections of patients with HD, see, for example, example 31 and FIG. 27-30, human-derived antibodies and their biotechnological derivatives are suitable for both the treatment and diagnosis of HD and the above-mentioned symptoms. Accordingly, according to one embodiment of the present invention, the antibodies of the invention, binding molecules having a binding specificity substantially identical to any of the above, polynucleotides, vectors, or cells of the present invention are used to prepare a pharmaceutical or diagnostic composition for prophylaxis and/or therapeutic treatment GD, in particular of NTT amyloidosis-related diseases and/or disorders, to monitor disease progression and/or response to treatment, and to diagnose diseases associated with NTT amyloidosis.

Treatment:

As used herein, the terms treat or treatment refer to both therapeutic treatment and prophylactic or prophylactic measures whose purpose is to prevent or slow (mitigate) an undesirable physiological change or disorder, such as the development of heart failure. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization (i.e., non-worsening) of a disease state, delay or slowing of disease progression, alleviation or attenuation of a disease state, and remission (partial or complete), detectable or undetectable. Treatment may also mean longer survival than expected without treatment. Those in need of treatment include those already suffering from the specified condition or disorder, as well as those predisposed to the specified condition or disorder, or the manifestations of the specified condition or disorder to be prevented.

Unless otherwise specified, the terms drug, drug, or medicament are used interchangeably herein and include, but are not limited to, all (A) articles, drugs, and compositions for internal or external use, and any substance or mixture of substances intended to for use in the diagnosis, cure, mitigation, treatment or prevention of disease in humans or other animals; and (B) products, drugs and compositions (not being food products) intended to affect the structure or any function of the body of humans or other animals; and (C) articles intended for use as a component of any article described in paragraphs. (A) and (B). The term drug, drug or medicament includes the complete formula of a composition intended for use in humans or other animals, containing one or more agents, compounds, substances or (chemical) compositions, as well as, in another context, other pharmaceutically inactive excipients, such as fillers, disintegrants, lubricants, lubricants, binders or providing ease of transport, disintegration, disaggregation, dissolution and bioavailability of the drug, drug or medicament at the intended target site in humans or other animals, for example, on the skin, in stomach or intestines. The terms agent, compound or substance are used interchangeably herein and include, more specifically, but not limited to, all pharmacologically active agents, i.e. agents that induce a desired biological or pharmacological effect, or agents that are examined or tested for the ability to induce such a possible pharmacological effect using the methods of the present invention.

By subject, individual, animal, patient or mammal is meant any subject, particularly a mammalian subject, such as a human patient, in need of diagnosis, prognosis, prophylaxis or therapy.

Pharmaceutical carriers:

Information regarding pharmaceutically acceptable carriers and methods of administration can be found in relevant publications known to one skilled in the art. Formulations of the pharmaceutical compositions of the present invention can be prepared in accordance with methods well known in the art; see for example: Remington: The Science and Practice of Pharmacy (2000), University of Sciences in Philadelphia, ISBN 0-683-306472; Vaccine Protocols, 2nd Edition, Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition, Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Formulations containing such carriers can be prepared using well known standard methods. Said pharmaceutical compositions may be administered to a subject at a suitable dose. The administration of suitable compositions can be accomplished in various ways. Examples include administering a composition containing a pharmaceutically acceptable carrier orally, intranasally, rectally, topically, intraperitoneally, intravenously, intramuscularly, subcutaneously, subdermally, transdermally, intrathecally, and intracranially. Aerosol formulations, such as nasal sprays, include purified aqueous or other solutions of the active agent with preservative and isotonic agents. Such formulations are preferably adjusted to isotonicity and pH values compatible with the mucous membranes of the nasal cavity. Pharmaceutical compositions for oral administration, such as single domain antibody molecules (eg, nanobodies™) and the like are also included in the present invention. Such oral formulations may be in the form of tablets, capsules, powder, liquid or semi-solid form. The tablet may contain a solid carrier such as gelatin or an adjuvant. Formulations for rectal or vaginal administration may be presented as a suppository in a suitable carrier; see also O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727-735. Further guidance regarding formulations suitable for administration by various routes can be found in: Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985) with appropriate updates. For a brief overview of drug delivery methods, see Langer, Science 249 (1990), 1527-1533.

II. Antibodies of the present invention

The present invention relates generally to human-derived antibodies to HTT and HTT-binding fragments thereof, preferably exhibiting the immunological binding characteristics and/or biological properties described for the antibodies illustrated in the examples section. In accordance with the present invention, human monoclonal antibodies specific for HTT were cloned from B cells of a population of healthy human subjects. However, in another embodiment of the present invention, human monoclonal antibodies to NTT can also be cloned from B cells of patients who exhibit symptoms of the disease and/or disorder associated with NTT amyloidosis.

In experiments carried out in accordance with the present invention, antibodies present in the conditioned medium of cultured human memory B cells were assessed for their ability to bind to HTT and more than 10 other proteins, including bovine serum albumin (BSA); see examples 8, 13, 18, 31 and 33. Only B cell supernatants capable of binding HTT protein, but not any of the other proteins in the screen, were selected for further analysis, including determination of antibody class and subclass light chains. Selected B cells were then processed for antibody cloning.

Briefly, the processing involved extraction of messenger RNA from selected B cells, reverse transcription by reverse transcription-PCR, amplification of antibody coding regions by PCR, cloning into plasmid vectors, and sequencing. Selected human antibodies were then synthesized and purified by recombinant expression in HEK293 or CHO cells and characterized for their ability to bind human HTT protein. Using a combination of various tests, for example, recombinant expression of antibodies in HEK293 or CHO cells and subsequent determination of the specificity of their binding to the human HTT protein, and the pronounced binding of pathologically mutated and/or aggregated forms of the human HTT protein,

- 26 046697 confirmed that for the first time human antibodies with high specificity for HTT have been cloned, which clearly recognize and selectively bind pathologically aggregated forms of the HTT protein. In some cases, mouse hybrid antibodies have also been prepared based on the variable domains of the human antibodies of the present invention.

Accordingly, the present invention relates generally to recombinant human-derived monoclonal antibodies to HTT and HTT-binding fragments thereof, synthetic and biotechnological derivatives and variants of these antibodies. According to one embodiment of the present invention, said antibody is capable of binding human HTT.

According to one embodiment of the present invention, the antibody specifically binds an epitope in the polyproline region of HTT, which contains the amino acid sequence PPPPPRRRR (NI-302.33C11; NI-302.44D7; NI-302.7A8; NI-302.3D8; NI-302.46C9) (SEQ ID No: 139, 151, 154, 158, 161), amino acid sequence PPPRRRR (NI-302.11H6, No.-302.18A1, NI-302.52C9 (SEQ ID No: 157, 159, 160), amino acid sequence PPPRRRRRRRR (NI-302.74 C11, NI-302.15F9, NI302.39G12, No.-302.11A4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6 (SEQ ID No: 146, 147, 148 , 149, 150, 152, 153, 155, 156), an epitope in the P-rich region that contains the amino acid sequence PQPPPQAQPL (NI-302.63F3 SEQ ID No: 140, NI-302.64E5 SEQ ID No: 200), amino acid sequence PPPQLPQPPP (NI-302.31F11, SEQ ID No: 141), amino acid sequence QAQPLLPQPQPPPPP (NI-302.2A2; SEQ ID No: 142) or amino acid sequence PPPQLPQPPPQAQPL (NI302.15D3; SEQ ID No: 143), epitope in the C-terminal region , which contains the amino acid sequence PPGPAVAEEPLHRP (NI-302.35C1, SEQ ID No: 145) or PPPGPAVAEEPLH (NI-302.72F10, SEQ ID No: 202), an epitope in the N-terminal region that contains the amino acid sequence KAFESLKSFQ (NI-NI- 302.15E8, SEQ ID No: 144) or an epitope in the P/Q rich region that contains the amino acid sequence QQQQQQQQQPPP (NI-302.7D8 SEQ ID No: 201); or conformational epitope.

In another embodiment, the present invention provides an anti-HTT antibody, or an antigen binding fragment, variant, or biotechnological derivative thereof, wherein said antibody specifically binds to the same epitope in the polyproline region of HTT as a reference antibody selected from the group consisting of NI- 302.33C11, NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-3O2.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, 3 02.78 H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9. Epitope mapping identified a sequence within the polyproline (poly-P) region of human HTT, including the amino acids PPPPPPPPPP (SEQ ID No: 146, 147, 148, 149, 150, 152, 153, 155, 156) as a unique linear epitope recognized antibodies NI302.74C11, NI-302.15F9, NI-302.39G12, NI-3O2.11A4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI302.78H12, NI-302.71F6 according to the present invention. In addition, epitope mapping identified a sequence within the polyproline region of human HTT, including the amino acids PPPPPPP (SEQ ID No: 139, 151, 154, 158, 161) as a unique linear epitope recognized by antibodies NI-302.33C11, NI-302.44D7 , NI-302.7A8, NI-302.3D8, NI-3O2.46C9 according to the present invention, and amino acids PPPPP (SEQ ID No: 157, 159, 160) as a unique linear epitope recognized by antibodies NI-3O2.11H6, NI- 3O2.18A1, NI-302.52C9 according to the present invention. Accordingly, according to one embodiment, the present invention provides an antibody wherein said antibody specifically binds to an epitope in the polyproline region of HTT that contains the amino acid sequence PPPPPRRRRPP (SEQ ID Nos: 146, 147, 148, 149, 150, 152, 153 , 155, 156), PPRRRRRR (SEQ ID No: 139, 151, 154, 158, 161) or RRPPRR (SEQ ID No: 157, 159, 160).

In one embodiment, the present invention provides an anti-HTT antibody, or an antigen binding fragment, variant, or biotechnological derivative thereof, wherein said antibody specifically binds to the same epitope in the P-rich region of HTT as a reference antibody selected from the group consisting of NI -302.63F3, NI-302.31F11, NI-302.2A2 and NI-302.15D3. Epitope mapping identified a sequence within the P-rich region of human HTT, including the amino acids PQPPPQAQPL (SEQ ID No: 140) as a unique linear epitope recognized by the antibody NI-302.63F3 according to the present invention, PPPQLPQPPP (SEQ ID No: 141) as a unique linear epitope recognized by the antibody NI-302.31F11 according to the present invention, PPPQLPQPPP (SEQ ID No: 141) as a unique linear epitope recognized by the antibody NI-302.31F11 according to the present invention, QAQPLLPQPQPPPPP (SEQ ID No: 142) as a unique linear epitope , recognized by the antibody NI-302.2A2, PPPQLPQPPPQAQPL (SEQ ID No: 143) as a unique linear epitope recognized by the antibody NI302.15D3, PQPPPQAQPL as a unique linear epitope recognized by the antibody NI3O2.64E5. Accordingly, according to one embodiment, the present invention provides an antibody, characterized in that the antibody specifically binds to an epitope in the P-rich region of HTT, which contains the amino acid sequence PQPPPQAQPL (SEQ ID No: 140), PPPQLPQPPP (SEQ ID No: 141), QAQPLLPQPQPPPPP (SEQ ID No: 142), PPPQLPQPPPQAQPL

- 27 046697 (SEQ ID No: 143).

In another embodiment, the present invention provides an anti-HTT antibody, or an antigen binding fragment, variant or biotechnological derivative thereof, characterized in that the antibody specifically binds to the same epitope in the polyglutamine/polyproline region of HTT as the reference antibody NI-302.7D8. Epitope mapping identified a sequence within the Q/P-rich region of human HTT comprising the amino acids QQQQQQQPPP (SEQ ID No: 201) as the unique linear epitope recognized by the NI-302.7D8 antibody of the present invention. Accordingly, according to one embodiment, the present invention provides an antibody, characterized in that the antibody specifically binds to an epitope in the polyglutamine/polyproline region of HTT, which contains the amino acid sequence QQQQQQQPPP (SEQ ID No: 201)

In one embodiment, the present invention provides an anti-HTT antibody, or an antigen-binding fragment, variant, or biotechnological derivative thereof, wherein said antibody specifically binds to the same epitope in the C-terminal region of HTT as a reference antibody selected from the group consisting of NI-302.35C1. Epitope mapping identified a sequence within the C-terminal region of human HTT comprising the amino acids PPGPAVAEEPLHRP (SEQ ID No: 145) as a unique linear epitope recognized by the NI-302.35C1 antibody of the present invention. Accordingly, according to one embodiment, the present invention provides an antibody, characterized in that the antibody specifically binds to an epitope in the C-terminal region of HTT, which contains the amino acid sequence PPGPAVAEEPLHRP (SEQ ID No: 145).

In a further embodiment, the present invention provides an anti-HTT antibody, or an antigen binding fragment, variant or biotechnological derivative thereof, wherein the antibody specifically binds to the same epitope in the C-terminal region of HTT as the reference antibody NI-302.72F10. Epitope mapping identified a sequence within the C-terminal region of human HTT comprising the amino acids PPPGPAVAEEPLH (SEQ ID No: 202) as a unique linear epitope recognized by the NI-302.72F10 antibody of the present invention. Accordingly, according to one embodiment, the present invention provides an antibody, characterized in that the antibody specifically binds to an epitope in the C-terminal region of HTT, which contains the amino acid sequence PPPGPAVAEEPLH (SEQ ID No: 202).

In another embodiment, the present invention provides an anti-HTT antibody, or an antigen binding fragment, variant, or biotechnological derivative thereof, wherein the antibody specifically binds to the same epitope in the N-terminal region of HTT as the reference antibody NI-3O2.15E8. Epitope mapping identified a sequence within the N-terminal region of human HTT comprising the amino acids KAFESLKSFQ (SEQ ID No: 144) as a unique linear epitope recognized by the NI-3O2.15E8 antibody of the present invention. Accordingly, according to one embodiment, the present invention provides an antibody, characterized in that the antibody specifically binds to an epitope in the N-terminal region of HTT, which contains the amino acid sequence KAFESLKSFQ (SEQ ID No: 144).

In one embodiment, the present invention provides an anti-HTT antibody, or an antigen-binding fragment, variant, or biotechnological derivative thereof, wherein said antibody specifically binds to the same exon 1-encoded HTT epitope as a reference antibody selected from the group consisting of NI -302.6N9, NI-302.4A6, NI-3O2.12H2, or NI-302.8M1 which have not been shown to bind linear HTT peptides encoded by exon 1, but bind aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamines repeats (HD21 and HD49) with high affinity and EC ₅₀ values in the subnanomolar range; see, for example, general information in Example 25 and FIG. 20. Accordingly, in one preferred embodiment, the antibody of the present invention specifically binds aggregated forms of HTT, in particular, protein aggregates derived from HTT encoded by exon 1, with an EC50 value of less than 1 nM, preferably less than 0.1 nM and most preferably less than 0.01 nM.

In addition, without reference to the original experimental studies, as shown in the examples and drawings, human monoclonal antibodies NI-302.33W1, NI-302.63F3, NI-302.35C1, NI-302.31F11, NI-302.6N9, NI-302.46C9 , NI-302.8F1, NI-302.74G11, NI-302.15F9, NI-302.39G12, NI302.11A4, NI-302.22H9, NI-302.44D7, NI-302.55D8, NI-302.7A8, NI-302.78H12, NI-302.71F6, No.-302.11H6, NI-302.3D8, and NI.3O2-64E5 and NI.302-72F10 to HTT according to the present invention are preferably characterized by specific binding to pathological mutated and/or aggregated HTT and significantly lower affinity for recognition of the physiological form of NTT, see, for example, examples 7, 13, 18 and Fig. 3, 7, 11, 21, 32. Thus, the present invention provides a set of human antibodies to HTT, characterized by binding characteristics suitable, in

- 28 046697 in particular, for diagnostic and therapeutic purposes. Accordingly, in one embodiment, the present invention provides antibodies that are capable of specifically binding to pathologically aggregated forms of HTT. However, in an alternative or additional embodiment, antibodies of the present invention that are capable of binding to the polyproline region or proline-rich region of HTT encoded by exon 1 can also be used for other purposes. In particular, these antibodies are not limited to HTT and can also bind to other targets, also identifying the polyproline tract or proline-rich region.

In one embodiment, an antibody of the present invention exhibits the binding characteristics of exemplary antibodies NI-302.33C11, NI-302.63F3, NI-302.35C1, NI.302-7D8, and NI.302-72F10 as described in the Examples section. The anti-HTT antibody of the present invention more preferably recognizes pathologically altered HTT, eg, mutated and/or aggregated HTT species and fragments thereof, than physiological HTT. Accordingly, in one embodiment, the antibody of the present invention does not substantially recognize physiological HTT species.

The term essentially does not recognize, as used herein to describe the binding affinity of a molecule from the group comprising an antibody, a fragment thereof, or a molecule that binds a specific target molecule, antigen, and/or target molecule and/or antigen in a particular conformation, means that the molecule from the above groups binds the specified molecule, antigen and/or conformational form with a binding affinity that is at least 2, 3, 4, 5, 6, 7, 8 or 9 times less than the binding affinity of a molecule from the above group for another molecule , antigen and/or conformation. Very often, the dissociation constant (KD) is used as a measure of binding affinity. Sometimes a specific assay, such as an ELISA, uses EC ₅₀ as a measure of binding affinity. Preferably, the term substantially does not recognize as used herein means that a molecule of the above group binds said molecule, antigen and/or conformer with an affinity of at least 10, 20, 50, 100, 1000 or 10,000 times less than the binding affinity of the molecule the above group with another molecule, antigen and/or conformer.

As described above, it is proposed that HTT aggregation in HD occurs as a result of elongation of the Htt polyglutamine tract encoded by exon 1. In particular, GD has been shown to mainly occur in patients with the number of glutamine residues in HTT exceeding a threshold of 35–40 residues. Accordingly, as shown in Example 3, aggregated and soluble HTT constructs encoded by exon 1 with 21, 35 or 49 polyglutamine repeats were generated to determine the ability of the anti-HTT antibodies of the present invention to specifically bind pathologically altered HTT.

The term HDX in the description below refers to the HTT constructs prepared in accordance with Example 3. In particular, X denotes the number of glutamine repeats (Q), for example, an HTT protein encoded by exon 1 with 21 polyglutamine repeats would be called HD21.

Using the constructs as described in the Examples section, the anti-HTT antibody of an additional or alternative embodiment of the present invention binds to pathological, disease-causing and/or mutated and/or aggregated forms of human HTT. In this context, binding affinities may be in the range indicated in the examples for antibodies NI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31F11, NI302.6N9, NI-302.46C9, NI-302.8F1, NI-302.74C11, NI-302.15F9, NI-302.39G12, No.-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.55D8, NI-302.7A8, NI-3O2.78H12, NI-302.71F6 , NI-3O2.11H6 and NI-302.3D8 in Figs. 3(A), 7(A), 11(A), 14(A) corresponding to FIG. 19, 20 and 31, i.e. correspond to half-maximal effective concentrations (EC50) of from about 1 pM to 250 nM, preferably an EC50 of from about 25 pM to 50 nM, most preferably an EC50 of from about 0.05 to 30 nM for aggregated human HD49-HTT and aggregated recombinant HD49-HTT, or EC ₅₀ , ranging from approximately 0.05 to 5 nM for aggregated human HD21-HTT and aggregated recombinant HD21-HTT.

In particular, an anti-HTT antibody, a binding fragment thereof, or a biotechnological derivative of said antibody has a binding affinity corresponding to an EC ₅₀ value of <20 nM, preferably <10 nM, and most preferably <1 nM for aggregated HD49 HTT; and/or being <40 nM, preferably <10 nM and most preferably <1 nM for HD21 HTT; see fig. 3, 7, 11, 19 and 31.

HTT aggregation associated with HD development is most often associated with polyglutamine (poly-Q) tracts containing >35 repeats. As shown in the present invention, anti-HTT antibodies as described herein exhibited high binding efficiency to BG tracts containing a significant number of repeats, see, for example, Examples 7, 13, 18, 31 and 33. Accordingly, according to one embodiment of the present of the invention, an anti-HTT antibody, an HTT-binding molecule, fragment, synthetic or biotechnological variant binds to an HTT extended beyond

- 29 046697 polyglutamine (Q) tract account. In a preferred embodiment, it binds to an HTT containing more than 35 repeats. According to a particular preferred embodiment of the present invention, the antibody binds to an HTT extended by a polyglutamine (Q) tract consisting of 49 (HD49) repeats, more than 35 repeats (HD35), and more than 21 repeats (HD21).

Moreover, the present invention also provides anti-HTT antibodies, HTT-binding molecules, fragments thereof, synthetic or biotechnological variants that bind to polyglutamine (poly-Q) tracts of less than 35 repeats (HD35). Accordingly, according to one embodiment of the present invention, said antibody, binding molecule, or variants thereof bind(s) to HTT containing normal polyglutamine tracts. In particular, the antibody is capable of binding HTT containing polyglutamine tracts of <35 repeats (HD35).

Some antibodies are able to bind a wide range of biomolecules, such as proteins. As will be understood by one skilled in the art, the term specific is used herein to mean that biomolecules other than HTT proteins or fragments thereof do not significantly bind to an antigen binding molecule, for example, one of the antibodies of the present invention. Preferably, the level of binding to a biomolecule other than HTT results in a binding affinity of no more than 20% or less, 10% or less, only 5% or less, only 2% or less, or only 1% or less (i.e. at least 5-, 10-, 20-, 50- or 100-fold lower affinity; any options other than those listed are also included) from affinity to HTT, respectively; see, for example, Fig. 20.

In one embodiment, the anti-HTT antibody of the present invention binds predominantly aggregated forms of HTT, and/or fragments, derivatives, fibrils and/or oligomers thereof. In another embodiment, the anti-HTT antibody of the present invention preferentially binds to both native HTT and pathologically mutated and/or aggregated forms of HTT.

According to a further embodiment of the present invention, an anti-HTT antibody or an HTT-binding fragment thereof, a synthetic or biotechnological derivative of the antibody is a bispecific antibody. Accordingly, an antibody of the present invention may be capable of recognizing at least two different epitopes on either the same or different antigens; see also above.

In one embodiment, at least one binding site/domain of the bispecific antibody specifically recognizes an epitope in the polyproline region of the HTT that contains the amino acid sequence PPPPPRRPP (NI-302.33C11; NI-302.44D7; NI-302.7A8; NI-302.3D8; NI302. 46C9) (SEQ ID No: 139, 151, 154, 158, 161), amino acid sequence PPPRRPP (NI-3O2.11H6, NI-3O2.18A1, NI-302.52C9 (SEQ ID No: 157, 159, 160), amino acid sequence PPPPPPPPPPP (NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.37a2, NI-302.55D8, NI302.78H12, NI-302.71F6 (SEQ ID No: 146, 147, 148, 149, 150, 152, 153, 155, 156), an epitope in the proline-rich region that contains the amino acid sequence PQPPPQAQPL (NI-302.63F3 SEQ ID No: 140, NI-3O2.64E5 SEQ ID No: 200), amino acid sequence PPPQLPQPPP (NI-302.31F11, SEQ ID No: 141), amino acid sequence QAQPLLPQPQPPPPP (NI-302.2A2; SEQ ID No: 142) or amino acid sequence PPPQLPQPPPQAQPL (NI302.15D3; SEQ ID No: 143), an epitope in the C-terminal region, which contains the amino acid sequence PPGPAVAEEPLHRP (NI302.35C1, SEQ ID No: 145) or PPPGPAVAEEPLH (NI-302.72F10, SEQ ID No: 202), an epitope in the N-terminal region, which contains the amino acid sequence KAFESLKSFQ (NI-302.15E8, SEQ ID No: 144), an epitope in a P/Q-rich region that contains the amino acid sequence QQQQQQQQQPPP (NI-302.7D8 SEQ ID No: 201), or a conformational epitope recognized by any of antibodies NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1.

As mentioned previously, the accumulation of polyglutamine (poly-Gln, poly-Q) HTT protein aggregates in the intranuclear inclusions of neurons is a hallmark of the progressive neurodegenerative disorder Huntington's disease (HD). Electron micrographs of these aggregates revealed fibrillar structures with a morphology similar to amyloid B fibrils in Alzheimer's disease, see for example Caughey et al., Trends Cell Biol. 7 (1997), 56-62 and Caputo et al., Arch. Biochem. Biophys. 292 (1992), 199-205, which suggests that in HD there is a degenerative process, mainly affecting striatal medium spiny neurons and cortical neurons, leading to dysfunction and subsequent loss of neurons, tissue damage due to excitotoxicity, mitochondrial damage, the formation of loose radicals, as well as possibly inflammatory mechanisms, including microglial activation and the progressive nature of further symptoms, are due to toxic amyloid fibrillogenesis. Accordingly, in one embodiment, an antibody of the present invention is suitable for treating Huntington's disease (HD) and its symptoms.

- 30 046697

To date, intracellularly expressed antibodies (intrabodies) have been described as therapeutic tools for HD that impair HTT function, see, for example, Ali et al., in Neurobiology of Huntington's Disease: Applications to Drug Discovery, Lo et al. , Chapter 10, CRC Press (2011). Although these intrabodies had a positive effect on HTT-induced aggregation and cell death in cell-based assays, see, for example, Khoshnan et al., Proc Natl Acad Sci USA. 99 (2002), 1002-1007, the method of their administration represents one of the disadvantages that reduces the therapeutic usefulness. In particular, viral vector-based gene therapy is a preferred method for delivering therapeutic intrabodies to the brain. However, the main disadvantage of this method of administration is, among other things, its high immunogenicity for the host. Accordingly, for therapeutic or diagnostic use, it is preferable to use non-viral routes involving other routes of administration. Antibodies of the present invention have been shown to reduce the loss of dendritic spine density when added to the culture medium, i.e. extracellular. Accordingly, the antibodies of the present invention, unlike previously described intrabodies, can be expected to be effective when using therapeutically preferred routes of administration. Accordingly, according to one embodiment of the present invention, the antibody is administered by subcutaneous injection (SC), intravenous injection (IV), intramuscular injection (IM), intraperitoneal (IP), intrathecal, bolus injection, wherein the range of action is not limited by the intracellular expression of the antibody.

As mentioned above and as shown in Example 24 and FIG. 17, the therapeutic utility of the antibodies of the present invention has been demonstrated. In particular, the anti-HTT antibodies of the present invention have been shown to be able to reduce the reduction in dendritic spine density. Accordingly, according to one embodiment of the present invention, an anti-HTT antibody, an HTT-binding fragment thereof, a synthetic or biotechnological derivative of the antibody provides a reduction in the decrease in spine density.

Moreover, the therapeutic utility of the antibodies of the present invention was demonstrated in Example 34 and shown in FIG. 34. In particular, the anti-HTT antibodies of the present invention have been shown to restore behavioral function during task-directed learning and improve motor function. Accordingly, according to one embodiment of the present invention, an anti-HTT antibody, an HTT-binding fragment thereof, a synthetic or biotechnological derivative of the antibody provides improved behavioral performance during targeted training to perform specific tasks and enhance sensorimotor abilities.

The present invention also provides an antibody or an antigen binding fragment, variant or derivative thereof containing an antigen binding domain identical to that of an antibody selected from the group consisting of NI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31 F11, NI-302.2A2, NI-302.6N9, NI-302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI302.37C12, NI-302.55D8 , NI-3O2.7A8, NI-302.78H12, NI-302.71F6, No.-302.11Н6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9, NI-302.15 E8, NI-302.15D3, NI-302.64E5, NI-302.7D8, NI302.72F10, NI-302.12H2, NI-302.8M1 and NI-3024A6.

The present invention also provides a number of examples of binding molecules, for example, antibodies and binding fragments thereof, recognizing the polyproline region of HTT, which may be characterized by the presence in the variable region, for example, in the binding domain, of at least one complementarity determining region (CDR) from the variable region VH and/or _VL containing any of the amino acid sequences shown in FIG. 1. The corresponding nucleotide sequences encoding the above-described variable regions are given in table. II below. Examples of sets of CDR regions of the VH and/or VL region amino acid sequences described above are shown in FIG. 1. However, as discussed below, it is well known to one skilled in the art that alternative or additional CDR variants may be used that have amino acid sequences different from those shown in FIG. 1, one, two, three or even more amino acids, in the case of CDR2 and CDR3. Accordingly, one embodiment provides an antibody of the present invention or an HTT-binding fragment thereof comprising in the variable region at least one complementarity determining region (CDR) according to FIG. 1, and/or one or more corresponding CDR regions including one or more amino acid substitutions.

The present invention also provides a number of examples of binding molecules, for example, antibodies and binding fragments thereof, recognizing the P-rich region of HTT, which may be characterized by the presence in the variable region, for example, in the binding domain, of at least one complementarity determining region (CDR) of the variable a VH and/or VL region containing any of the amino acid sequences shown in FIG. 1. The corresponding nucleotide sequences encoding the above-described variable regions are given in table. III below. Examples of sets of CDR regions of the above-described VH region amino acid sequences

- 31 046697 and/or VL are shown in Fig. 1. However, as discussed below, it is well known to one skilled in the art that alternative or additional CDR variants may be used that have amino acid sequences different from those shown in FIG. 1, one, two, three or even more amino acids, in the case of CDR2 and CDR3. Accordingly, one embodiment provides an antibody of the present invention, or an HTT-binding fragment thereof, comprising in the variable region at least one complementarity determining region (CDR) as shown in FIG. 1, and/or one or more corresponding CDR regions including one or more amino acid substitutions.

The present invention also provides a number of examples of binding molecules, for example, antibodies and binding fragments thereof, recognizing the C-terminal region of HTT, which may be characterized by the presence in the variable region, for example, the binding domain of at least one complementarity determining region (CDR) of the VH variable region and/or VL containing any of the amino acid sequences shown in FIG. 1. The corresponding nucleotide sequences encoding the above-described variable regions are given in table. IV below. Examples of sets of CDR regions of the V _H and/or V _L region amino acid sequences described above are shown in FIG. 1. However, as discussed below, it is well known to one skilled in the art that alternative or additional CDR variants may be used that have amino acid sequences different from those shown in FIG. 1, one, two, three or even more amino acids, in the case of CDR2 and CDR3. Accordingly, one embodiment provides an antibody of the present invention, or an HTT-binding fragment thereof, comprising in the variable region at least one complementarity determining region (CDR) as shown in FIG. 1, and/or one or more corresponding CDR regions including one or more amino acid substitutions.

The present invention also provides a number of examples of binding molecules, for example, antibodies and binding fragments thereof, recognizing the N-terminal region of HTT, which may be characterized by the presence in the variable region, for example, in the binding domain, of at least one complementarity determining region (CDR) of a variable region V _H and/or V _L containing any of the amino acid sequences shown in FIG. 1. The corresponding nucleotide sequences encoding the above-described variable regions are given in table. VI below. Examples of sets of CDR regions of the V _H and/or V _L region amino acid sequences described above are shown in FIG. 1. However, as discussed below, it is well known to one skilled in the art that alternative or additional CDR variants may be used that have amino acid sequences different from those shown in FIG. 1, one, two, three or even more amino acids, in the case of CDR2 and CDR3. Accordingly, one embodiment provides an antibody of the present invention, or an HTT-binding fragment thereof, comprising in the variable region at least one complementarity determining region (CDR) as shown in FIG. 1, and/or one or more corresponding CDR regions including one or more amino acid substitutions.

In one embodiment, an antibody of the present invention is any of the antibodies comprising the V _H and/or V _L region amino acid sequence of FIG. 1, or a corresponding V _H and/or V _L region including one or more amino acid substitutions. Preferably, the antibody of the present invention is characterized by maintaining cognate pairing of heavy and light chains in accordance with that found in a human B cell.

In a further embodiment of the present invention, the anti-HTT antibody, HTT-binding fragment, synthetic or biotechnological variant thereof can be optimized to provide desired target binding affinity and pharmacokinetic properties. Accordingly, at least one amino acid in the CDR or variable region subject to modifications selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, isoaspartate formation and/or unpaired cysteine is replaced with a mutated amino acid not containing such change; or at least one carbohydrate moiety is removed from said antibody or chemically or enzymatically added to said antibody. Examples of amino acid optimization can be found in Table. VII, which presents antibodies with primer-induced changes. Additional modifications to optimize antibody performance are described in Gavel et al., Protein Engineering 3 (1990), 433-442 and Helenius et al., Annu. Rev. Biochem. 73 (2004), 1019-1049.

Alternatively, an antibody of the present invention is an antibody or antigen binding fragment, derivative or variant thereof that competes for HTT binding with at least one of the VH and/or VL region containing antibodies of FIG. 1.

An antibody containing at least one VH and/or VL region according to FIG. 1 competing for binding to HTT can be further examined using a dot blot and/or filter delay assay as described in Examples 6, 13, 18, 31 and/or 32. Accordingly, in one embodiment of the present invention, said antibody binds to HTT, preferably HTT extended by a 49-repeat polyglutamine tract (HD49), as assessed using dot blot and/or filter delay techniques.

The experimental results shown in Fig. 3, 7, 11, 21, 22, and also figs. 32 and 33, and Examples 6, 7, 13, 18, 26 and 32, suggest that some of the anti-HTT antibodies of the present invention preferentially bind to disease-causing mutated and/or aggregated forms of human HTT rather than to other proteins involved in the formation of amyloid. Accordingly, in one embodiment, the antibody of the present invention preferentially recognizes mutated and/or aggregated HTT, and/or fragment and/or derivatives thereof, rather than other proteins involved in amyloid formation.

According to one embodiment of the present invention, an anti-HTT antibody, its HTT-binding fragment, a synthetic or biotechnological derivative of the antibody more preferentially recognizes mutated, aggregated and/or soluble forms of HTT than physiological HTT.

The antibody of the present invention may be a human antibody, particularly for therapeutic use. Alternatively, the antibody of the present invention is a rodent antibody containing rodent antibody elements or a rodent/human hybrid antibody, preferably a mouse antibody containing mouse antibody elements or a mouse/human hybrid antibody; or a rat antibody containing rat antibody elements or a rat/human hybrid antibody, particularly suitable for use in diagnostic methods and animal studies. In one embodiment, the antibody of the present invention is a rodent/human hybrid antibody or an antibody containing rodent antibody elements. Moreover, according to one embodiment, the hybrid antibody of the present invention, i.e. containing human antibody variable domains and common mouse light and heavy chain constant domains, binds with high affinity to human HTT. Preferably, the binding affinity of the hybrid antibodies is similar to the binding affinity of the human counterparts.

In one embodiment, the antibody of the present invention is obtained from cultured monoclonal or oligoclonal cultures of B cells; the culture supernatant containing antibodies produced by the specified B cells is screened for the presence of antibodies to HTT and their affinity. The screening method includes screening for binding of native monomers, fibrillar or non-fibrillar aggregates, such as hHTT oligomers derived from a synthetic full-length hHTT peptide, or, for example, purified from human plasma or obtained by recombinant expression.

As described above, due to its synthesis during the immune response in humans, the human monoclonal antibody of the present invention recognizes epitopes, particularly those associated with pathology, that may not be accessible or less immunogenic when using immunization to produce, for example, mouse monoclonal antibodies, and screening phage libraries. in vitro display, respectively. Thus, it is reasonable to assume that the epitope of the human anti-HTT antibody of the present invention is unique, and there are no other antibodies capable of binding the epitope recognized by the human monoclonal antibody of the present invention. The uniqueness of the antibody of the present invention is further confirmed by the fact that, as shown in FIG. 19, 20, 24, 27-29, the antibodies of the present invention bind specific epitopes of mutated and/or aggregated forms of HTT, which, as noted above, are particularly associated with pathology, and conventional methods for producing antibodies, such as immunization or screening libraries in vitro may not be suitable for obtaining these antibodies.

Accordingly, in one embodiment, the present invention also generally covers anti-HTT antibodies and HTT-binding molecules that compete with the human monoclonal antibody of the present invention for specific binding to HTT. More specifically, the present invention provides an antibody or antigen binding fragment, variant or derivative thereof, characterized in that said antibody specifically binds to the same epitope in the polyproline region of the HTT as a reference antibody selected from the group consisting of NI -302.33C11, NI-302.74C11, NI-302.15F9, NI-302.39G12, No.-302.11A4, NI-302.22H9, NI-302.44D7, NI302.37C12, NI-302.55D8, NI-302.7A8, NI- 302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.78H12 and NI-302.46C9. Moreover, in one embodiment, the present invention more specifically provides an antibody or antigen binding fragment, variant or derivative thereof, characterized in that said antibody specifically binds to the same epitope in the proline-rich region of the HTT as a reference antibody , selected from the group consisting of NI-302.63F3, NI-302.31F11, NI-302.2A2, NI-302.15D3 and/or NI-302.64E5. In another embodiment, the present invention provides an antibody, or an antigen binding fragment, variant or derivative thereof, that binds to the same epitope in the C-terminal region of the HTT as a reference antibody selected from the group consisting of from NI-302.35C1 and/or NI.302-72F10. In a further embodiment, the present invention provides an antibody, or an antigen binding fragment, variant or derivative thereof, that binds to the same epitope in the N-terminal region of the HTT as the reference antibody selected from the group consisting of NI-3O2.15E8. In another embodiment, the present invention provides an antibody, or an antigen binding fragment, variant or derivative thereof, that binds to the same HTT epitope as a reference antibody selected from the group consisting of NI-302.6N9 , NI-320.12H2, NI-302.8M1 and/or NI-302.4A6. In one embodiment, the present invention provides an antibody, or an antigen binding fragment, variant or derivative thereof, that binds to the same HTT epitope as the reference antibody NI302.7D8. In a preferred embodiment, the present invention also generally relates to anti-HTT antibodies and HTT-binding molecules that compete with the human monoclonal antibody of the present invention for specific binding to mutated and/or aggregated HTT species or fragments thereof, as shown in Examples 7, 13, 18, 31 and 33, as well as in FIG. 7, 13, 19 and 31. Accordingly, the present invention also more particularly relates to an antibody or an antigen binding fragment, variant or derivative thereof, wherein said antibody specifically binds to the same epitope in the polyproline region of mutated and/or aggregated HTT species or fragments thereof , as the reference antibody selected from the group consisting of No.-302.74C11, NI-302.15F9, NI-302.39G12, NI-3O2.11A4, NI-302.22H9, NI-302.37C12, NI-302.55D8, NI302. 78H12, NI-302.71F6, NI-302.33C11, NI-302.44D7, NI-302.7A8, NI-302.3D8, NI-302.46C9, No.-302.11Н6, NI-302.18A1, NI-302.52C9 and/or NI -302.8F1. Moreover, in one embodiment, the present invention more specifically provides an antibody or an antigen binding fragment, variant or derivative thereof, wherein said antibody specifically binds to the same epitope in the proline-rich region of mutated and/or aggregated HTT species or fragments thereof, such that and a reference antibody selected from the group consisting of NI-302.63F3, NI-302.31F11, NI302.2A2, NI-302.15D3 and/or NI-302.64E5. In another embodiment, the present invention provides an antibody, or an antigen binding fragment, variant or derivative thereof, that binds to the same epitope in the C-terminal region of mutated and/or aggregated HTT species or fragments thereof, such that and a reference antibody selected from the group consisting of NI302.35C1 and/or NI.302-72F10. In a further embodiment, the present invention provides an antibody or an antigen binding fragment, variant or derivative thereof that binds to the same epitope in the N-terminal region of mutated and/or aggregated HTT species or fragments thereof, such that and a reference antibody selected from the group consisting of NI302.15E8. In another embodiment, the present invention provides an antibody or antigen binding fragment, variant or derivative thereof that binds to the same HTT epitope as a reference antibody selected from the group consisting of NI-302.6N9 , NI320.12H2, NI-302.8M1 and/or NI-302.4A6. In one embodiment, the present invention provides an antibody, or antigen binding fragment, variant, or biotechnological derivative thereof, that binds to the same HTT epitope as the reference antibody NI302.7D8.

Competition between antibodies is determined by an assay in which the immunoglobulin being tested inhibits the specific binding of a reference antibody to a common antigen, such as HTT. Numerous types of competitive binding assays are known, for example: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme-linked immunosorbent assay (ELISA), competitive sandwich assay; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; solid-phase direct ELISA with biotin and avidin; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 and Cheung et al., Virology 176 (1990), 546-552; direct solid-phase assay with labeling, direct solid-phase sandwich assay with labeling; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); direct solid-phase RIA with I ¹²⁵ labeling; see Morel et al., Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82. Typically, such an assay involves the use of purified HTT or mutated and/or aggregated HTT, for example, its oligomers and/or fibrils, bound to a solid surface or cells bearing any of the specified, unlabeled experimental immunoglobulin and labeled reference immunoglobulin, i.e. e. human monoclonal antibody according to the present invention. Competitive inhibition is measured by determining the amount of label bound to a solid surface or cells in the presence of an experimental immunoglobulin. As a rule, experimental immunoglobulin is present in excess. Preferably, the competitive binding assay is performed under conditions similar to those described for the ELISA in the examples. Antibodies identified by competition analysis (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope located close enough to the epitope bound by the reference antibody to cause steric hindrance. Typically, if a competing antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 50 or 75%. Thus, the present invention also relates to an antibody or antigen binding fragment, variant or derivative thereof, from

- 34 046697 characterized in that the specified antibody competitively inhibits binding to the HTT of a reference antibody selected from the group consisting of NI-302.33C11, NI-302.63F3, NI-302.35C1, NI-302.31F11, NI-302.2A2, NI- 302.6N9, NI-302.74C11, NI-302.15F9, NI-302.39G12, No.-302.11A4, NI-302.22H9, NI302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, NI-3OO2 .78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9, NI-302.15E8, NI-302.64E5, NI- 302.7D8, NI302.72F10, NI-302.12H2, NI-3O2.8M1 and/or NI-302.4A6.

The present invention also provides an antibody or an antigen-binding fragment, variant or derivative thereof, characterized in that said antibody competitively inhibits binding to mutated and/or aggregated HTT species or fragments thereof of a reference antibody selected from the group consisting of NI-302.74C11, NI -302.15F9, NI-302.39G12, NI-3O2.11A4, NI302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6, NI-302.33C11, NI-302.44D7, 302 .7A8, NI-302.3D8, NI-302.46C9, No.-302.11Н6, No.-302.18А1, NI-302.52C9, NI-302.8F1, NI-302.63F3, NI302.31F11, NI-302.2A2, NI302.15D3 , NI-302.35C1, NI-302.6N9, NI-302.7D8 and/or NI-302.72F10.

In a preferred embodiment of said antibody, the binding of the antibody, its binding fragment, synthetic or biotechnological variant to HTT, preferably HTT extended by a polyglutamine tract of 49 (HD49) repeats, can be measured using a dot blot assay and/or or filter delays as described in the Examples section, in particular 7, 13, 18, 31 and/or 33.

In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), wherein at least one of the VH-CDR regions of said heavy chain variable region or at least two VH regions The -CDR of said heavy chain variable region is at least 80, 85, 90 or 95% identical to the amino acid sequences of the reference heavy chain VH-CDR1, VH-CDR2 or VH-CDR3 from the antibodies as described herein. Alternatively, the VH-CDR1, VH-CDR2 and VH-CDR3 regions of said VH are at least 80, 85, 90 or 95% identical to the amino acid sequences of the reference heavy chain VH-CDR1, VH-CDR2 and VH-CDR3 from the antibodies as described in this document. Accordingly, in this embodiment, the heavy chain variable region of the present invention comprises VH-CDR1, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in FIG. 1, respectively. In fig. 1 represents VH-CDR regions defined by the Kabat system, however other CDR definitions, such as VH-CDR regions defined by the Chothia system, are also included in the present invention and can be readily identified by one skilled in the art using the data provided. in fig. 1.

In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions contain polypeptide sequences identical to the VH-CDR1 groups , VH-CDR2 and VH-CDR3 shown in FIG. 1 respectively. In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (V _H ), wherein the V _H -CDR1, V _H -CDR2, and V _H -CDR3 regions contain identical polypeptide sequences groups VH-CDR1, VH-CDR2 and VH-CDR3 shown in FIG. 1, respectively, except for one, two, three, four, five or six amino acid substitutions in any V _H -CDR. In certain embodiments, these amino acid substitutions are conservative.

In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), wherein at least one of the VL-CDR regions of said light chain variable region or at least two VL regions The -CDR of said light chain variable region is at least 80, 85, 90 or 95% identical to the amino acid sequences of the reference light chain V _L -CDR1, V _L -CDR2 or V _L -CDR3 from the antibodies as described herein. Alternatively, the VL-CDR1, VL-CDR2 and VL-CDR3 regions of said VL are at least 80, 85, 90 or 95% identical to the amino acid sequences of the reference light chain V _L -CDR1, V _L -CDR2 and V _L -CDR3 from antibodies as described herein. Accordingly, in this embodiment, the light chain variable region of the present invention comprises VL-CDR1, VL-CDR2 and VL-CDR3 polypeptide sequences related to the polypeptides shown in FIG. 1 respectively. In fig. 1 shows VL-CDR regions defined according to the Kabat system, however, other CDR definitions, such as VL-CDR regions defined according to the Chothia system, are also included in the present invention.

In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions contain polypeptide sequences identical to the VL- groups CDR1, VL-CDR2 and VL-CDR3 shown in FIG. 1, respectively. In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions contain identical polypeptide sequences groups VL-CDR1, VL-CDR2 and VL-CDR3 shown in FIG. 1, respectively, excluding one, two, three, four, five or six amino acid substitutions in any VL-CDR. In certain embodiments, these amino acid substitutions are conservative.

The immunoglobulin or the cDNA encoding it may be further modified. Accordingly, in a further embodiment, the method of the present invention includes any of the steps of producing a hybrid antibody containing mouse antibody elements, a single chain antibody, a Fab fragment, a bispecific antibody, a hybrid antibody, a tagged antibody, or an analogue of any of the above. Appropriate methods are known to one skilled in the art and are described, for example, in Harlow and Lane Antibodies, A Laboratory Manual, CSH Press, Cold Spring Harbor (1988). If derivatives of these antibodies are produced using phage display techniques, surface plasmon resonance can be used, as in the BIAcore system, to enhance the effectiveness of phage antibodies that bind to the same epitope as any of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of hybrid antibodies is described, for example, in international application WO 89/09622. Methods for producing humanized antibodies are described, for example, in European patent application EP-A1 0239400 and international application WO 90/07861. Additional sources of antibodies for use in accordance with the present invention are so-called xenogeneic antibodies. The general principle for the production of xenogeneic antibodies, such as human-like antibodies in mice, is described, for example, in international applications WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. As described above, the antibody of the present invention may exist in various forms other than complete antibodies; including, for example, in the form of Fv, Fab and F(ab)2, as well as single chains; see for example international application WO 88/09344. According to one embodiment, there is accordingly provided an antibody of the present invention selected from the group consisting of single chain Fv fragment (scFv), F(ab') fragment, F(ab) fragment and F(ab') ₂ fragment.

The antibodies of the present invention or their corresponding immunoglobulin chain(s) can be further modified using standard techniques known in the art, for example, by deleting, inserting, substituting, adding and/or recombining amino acid(s), and/or any other modification(s) known in the art, alone or in combination. Methods for introducing such modifications into a DNA sequence corresponding to the amino acid sequence of an immunoglobulin chain are well known to one skilled in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Modifications to an antibody of the present invention include chemical and/or enzymatic derivatization of one or more of its constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and attachment of fragments of carbohydrates or lipids, cofactors, etc. Likewise, the present invention covers the production of fusion proteins that contain the disclosed antibody or any fragment thereof at the amino terminus linked to a heterologous molecule, such as an immunostimulatory ligand, at the carboxyl terminus; relevant technical information can be found, for example, in international application WO 00/30680.

The antibodies of the present invention may also include additional modifications to optimize their therapeutic potential. These modifications include, but are not limited to, modifications to the amino acid sequence of the antibody (eg, variable regions) and post-translational modifications. Post-translational modifications (PTMs) are chemical modifications that play a key role in functional proteomics as they regulate activity, localization, and interactions with other cellular molecules such as proteins, nucleic acids, lipids, and cofactors. Accordingly, optimization of antibodies can provide a number of advantages, such as increased storage stability, as well as a pharmacokinetic and/or pharmacodynamic profile, e.g., in vivo or in vitro antibody circulation time, improved solubility, stability, increased target affinity, reduced dissociation rate, improved effector function of the constant region (Fc region) and safety profile of the antibody, for example, reduced immunogenicity or reduced susceptibility to post-translational modifications, as shown, for example, in Igawa et al., MAbs 3 (2011), 24352. Accordingly, according to one In an embodiment of the present invention, the anti-HTT antibody, its HTT-binding fragment, synthetic or biotechnological variant may be optimized such that at least one amino acid in the CDR region or variable region is subject to modifications, which include, but are not limited to, acetylation , acylation, ADP-ribosylation, amidation, deamidation, covalent addition

- 36 046697 flavin, covalent addition of a heme fragment, covalent addition of a nucleotide or nucleotide derivative, covalent addition of a lipid or lipid derivative, covalent addition of phosphatidylinositol, cross-linking, cyclization, formation of disulfide bonds, isomerization, demethylation, formation of covalent cross-links, formation of cysteine, formation pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, hydrolysis, iodination, methylation, myristiolation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, tRNA-mediated addition of amino acids to proteins, e.g. , arginylation and ubiquitination (see, for example, Creighton, Proteins: Structures and Molecular Properties, 2nd eds., Freeman and Co., N.Y., 1992; Posttranslational Covalent Modification of Proteins, Johnson, eds., Academic Press, New York, 1983 ; Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY. Acad. Sei. 663 (1992) 48-62), is replaced by a mutated amino acid that does not exhibit such a change; or at least one carbohydrate moiety is removed from said antibody or chemically or enzymatically added to said antibody. In a preferred embodiment, said modifications are selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, isoaspartate formation and/or unpaired cysteine. Additional modification to optimize the utility of anti-HTT antibodies or HTT-binding molecules as therapeutic agents is well known in the art and is described, for example, in Igawa et al., MAbs 3 (2011), 243-52, the contents of which are included in this document. The addition or removal of carbohydrate moieties can be achieved chemically or enzymatically and is described in detail in, for example, Berg et al. Biochemistry 5th eds W H Freeman, New York 2002; WO 87/05330; Aplin et al., CRC Crit. Rev. Biochem., 22 (1981), 259-306; Hakimuddin et al., Arch. Biochem. Biophys., 259 (1987), 10-52; Edge et al., Anal. Biochem., 118 (1981), 131; Thotakura et al., Meth. Enzymol. 138. (1987), 350.

Also covered by the present invention are peptides comprising a binding molecule as described above, for example comprising the CDR3 region of the variable region of any of said antibodies, in particular the heavy chain CDR3, since it has often been observed that the heavy chain CDR3 (HCDR3) represents is an area characterized by a greater degree of variability and predominant participation in the interaction of antigen with antibody. Such peptides can be readily synthesized or recombinantly produced to produce a binding agent suitable for use in the present invention. Such methods are well known to those skilled in the art. Peptides can be synthesized, for example, using automated commercially available peptide synthesizers. These peptides can also be produced using recombinant techniques by incorporating DNA to express said peptide into an expression vector and transforming cells with said expression vector to produce said peptide.

Thus, the present invention relates to any binding molecule, for example an antibody or binding fragment thereof, directed to antibodies to HTT and/or antibodies capable of binding mutated and/or aggregated HTT species and/or fragments thereof according to the present invention, and demonstrating the mentioned qualities, i.e. in particular, recognizing HTT and/or mutated and/or aggregated HTT species, and/or fragments thereof. Such antibodies and binding molecules can be tested for binding specificity and affinity using ELISA and immunohistochemistry as described herein, see, for example, the Examples. Testing of these characteristics of antibodies and binding molecules can also be carried out using Western blotting.

In an alternative to producing immunoglobulins directly from a culture of B cells or memory B cells, the cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation. The rearranged antibody genes can be reverse transcribed from the appropriate mRNAs to produce cDNAs. If necessary, the heavy chain constant region can be replaced with a region of a different isotype or completely removed. Variable regions can be connected to encode single-chain Fv regions. Multiple Fv regions can be linked to provide binding ability to more than one target; or combinations of hybrid heavy and light chains may be used. Given the available genetic material, designing analogs as described above that retain the ability to bind the desired target is straightforward. Methods for cloning antibody variable regions and producing recombinant antibodies are known to one skilled in the art and are described, for example, in: Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

After obtaining suitable genetic material and, if necessary, modifying it to encode the analogue, the coding sequences, including those encoding at least the variable regions of the heavy and light chains, can be integrated into expression systems contained in vectors with which standard recombinant host cells.

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Various such host cells can be used; however, mammalian cells are preferred for efficient processing. Exemplary mammalian cell lines suitable for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or

NSO.

The antibody or analogue thereof is then produced by culturing the modified recombinant host under culture conditions suitable for growth of said host cells and expression of coding sequences. The antibodies are then recovered by isolation from the culture. Expression systems are preferably designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.

As described above, the present invention also provides a polynucleotide encoding an antibody or equivalent binding molecule of the present invention, in the case of an antibody, preferably at least an immunoglobulin chain variable region of the antibody as described above. Typically, said variable region encoded by the polynucleotide contains at least one complementarity determining region (CDR) of the VH and/or VL variable region of said antibody.

It will be apparent to one skilled in the art that the variable domain of an antibody containing the above-described variable domain can be used to construct other polypeptides or antibodies having the desired specificity and biological function. Accordingly, the present invention also covers polypeptides and antibodies containing at least one CDR region of the variable domain described above and providing the advantage of binding characteristics substantially identical or similar to those of the antibody described in the accompanying examples. One skilled in the art will know that binding affinity can be increased by making amino acid substitutions within the CDRs or hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) that partially overlap the CDR regions as defined by Kabat; see, for example, Riechmann, et al., Nature 332 (1988), 323-327. Accordingly, the present invention also relates to antibodies in which one or more of said CDR regions contain one or more substitutions, preferably no more than two amino acid substitutions. Preferably, one or both immunoglobulin chains of the antibody of the present invention comprise two or all three CDR variable regions according to FIG. 1.

Binding molecules, eg antibodies or antigen binding fragments, synthetic or biotechnological variants or derivatives thereof of the present invention, as known to those skilled in the art, may contain a constant region that mediates one or more effector functions. For example, binding of complement component C1 to the constant region of an antibody can activate the complement system. Complement activation is important for the opsonization and lysis of cellular pathogens. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, antibodies bind to receptors on various cells through the Fc region, wherein the Fc receptor binding site in the Fc region of the antibody binds to the Fc receptor (FcR) on the cell. There are a number of Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Antibody binding to F receptors on cell surfaces triggers a variety of important biological reactions, including uptake and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin synthesis.

Accordingly, certain embodiments of the present invention include an antibody or an antigen binding fragment, variant or derivative thereof, wherein at least a portion of one or more constant region domains has been deleted or otherwise altered to provide desired biochemical characteristics, e.g., reduced effector functions, ability to non-covalent dimerization, increased ability to localize to the site of HTT aggregation and deposition, reduced serum half-life or increased serum half-life compared to intact intact antibody of approximately the same immunogenicity. For example, some antibodies for use in the diagnostic and therapeutic methods described herein are domain-deleted antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain, but the chain lacks at least a portion of one or more heavy chain domains. Thus, in certain antibodies, one domain of the constant region of the modified antibody is completely removed, for example, the CH2 domain is completely or partially removed. In other embodiments, certain antibodies for use in the diagnostic and therapeutic methods described herein comprise a constant region, such as an IgG heavy chain constant region, that is altered

- 38 046697 for elimination of glycosylation; such antibodies are referred to herein as aglycosylated or Agly antibodies. Such Agly antibodies can be produced enzymatically, as well as by engineering consensus glycosylation site(s) in the constant region. Without being bound by theory, it is believed that Agly antibodies may have an improved safety and stability profile in vivo. Methods for preparing aglycosylated antibodies having the desired effector function are described, for example, in international application WO 2005/018572, which is incorporated herein by reference in its entirety.

In certain antibodies or antigen binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated to reduce effector function using techniques known in the art. For example, deletion or inactivation (by point mutations or other means) of the constant region domain may reduce Fc receptor binding of the circulating modified antibody, promoting localization to regions where HTT is present. In other cases, modifications to the constant region in accordance with the present invention may limit complement fixation and, accordingly, reduce the serum half-life and nonspecific binding of the conjugated cytotoxin. Other constant region modifications may be used to modify disulfide bonds or oligosaccharide moieties to provide improved localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of these modifications, such as HTT localization, biodistribution and serum half-life, can be easily measured and quantified using well-known immunological techniques without undue experimentation.

In certain antibodies or antigen binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated or replaced with alternative protein sequences to enhance cellular uptake of the antibodies by, for example, enhancing receptor-mediated endocytosis of antibodies through FcY receptors, LRPs or receptors Thy1, or through superantibody technology, which is reported to allow the introduction of antibodies into living cells without damaging them (Expert Opin. Biol. Ther. (2005), 237-241). For example, fusion proteins of the antibody binding region and cognate protein ligands of cell surface receptors or bi- or multispecific antibodies with specific sequences that bind to HTT as well as cell surface receptors can be constructed using techniques known in the art.

In certain antibodies or antigen binding fragments, variants or derivatives thereof described herein, the Fc portion may be mutated or replaced by alternative protein sequences, or the antibody may be chemically modified to increase the ability to penetrate the blood-brain barrier.

Modified forms of antibodies or antigen binding fragments, variants or derivatives thereof according to the present invention can be obtained from full-length precursor antibodies or parent antibodies using techniques known in the art. Examples of techniques are described in detail in this document. Antibodies or antigen binding fragments, variants or derivatives thereof of the present invention can be prepared or produced using techniques known in the art. In certain embodiments, antibody molecules or fragments thereof are produced recombinantly, i.e. using recombinant DNA technology. Examples of techniques for producing antibody molecules or fragments thereof are described in detail in various sections of this document.

Antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention also include derivatives modified, for example, by covalently attaching a molecule of any type to the antibody that does not prevent the antibody from specifically binding to a cognate epitope. For example, but without limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, and the like. Any of numerous chemical modifications can be accomplished using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, said derivative may contain one or more non-classical amino acids.

In specific preferred embodiments, the antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention do not elicit a damaging immune response in the animal being treated, such as a human. In certain embodiments, the binding molecules, e.g., antibodies or antigen-binding fragments thereof, of the present invention are derived from a patient, e.g., a human patient, and are subsequently used in the same species from which they are derived, e.g., a human, such that

- 39 046697 mitigates or minimizes the occurrence of damaging immune reactions.

Deimmunization may additionally be used to reduce the immunogenicity of the antibody. As used herein, the term deimmunization includes alteration of an antibody with modification of T cell epitopes; see, for example, international applications WO 98/52976 and WO 00/34317. For example, the VH and VL sequences of the parent antibody are analyzed and a human T cell epitope is mapped from each V region, identifying the location of the epitopes relative to complementarity determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify amino acid substitutions with little risk of altering the activity of the resulting antibody. A number of alternative VH and VL sequences are constructed containing combinations of amino acid substitutions; these sequences are then inserted into a series of binding polypeptides, for example, HTT-specific antibodies or immunospecific fragments thereof, for use in diagnostic and therapeutic methods as described herein, and then tested for functionality. Typically, 12 to 24 antibody variants are obtained and tested. The complete heavy and light chain genes containing the modified human V regions and C regions are then cloned into expression vectors and the resulting plasmids are introduced into cell lines to produce the complete antibody. These antibodies are then compared in appropriate biochemical and biological assays, and the best option is identified.

Monoclonal antibodies can be produced using a wide variety of techniques known in the art, including the use of hybridomas, recombinant technology and phage display technology, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and described, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., 563-681 (1981), these references are incorporated herein by reference in their entirety. The term monoclonal antibody as used herein is not limited to antibodies produced using hybridoma technology. The term monoclonal antibody refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and not to the method of its preparation. Accordingly, the term monoclonal antibody is not limited to antibodies produced using hybridoma technology. In certain embodiments, the antibodies of the present invention are derived from human B cells that have been immortalized by transformation with the Epstein-Barr virus as described herein.

In a well-known method for producing hybridomas (Kohler et al., Nature 256 (1975), 495), relatively short-lived or mortal lymphocytes of a mammal, for example B cells, derived from a human subject as described herein, are combined with an immortalized tumor cell line (for example, a myeloma cell line), respectively, producing hybrid cells or hybridomas that are immortalized and capable of producing a genetically encoded B-cell antibody. The resulting hybrids are separated into genetic monostrains by selecting, breeding, and repeatedly cultivating each of the individual strains containing specific genes to produce a single antibody. They produce antibodies of the same type to the desired antigen, which, due to their homogeneous genetic origin, are called monoclonal.

The hybridoma cells suitably prepared are seeded and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-hybrid myeloma parent cells. Those skilled in the art will appreciate that reagents, cell lines, and media for the production, selection, and cultivation of hybridomas are commercially available from a number of sources; standardized protocols are well known. As a rule, the culture medium where hybridoma cells are cultivated is analyzed for the presence of synthesized monoclonal antibodies to the desired antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined using in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) as described herein. Once hybridoma cells are identified that produce antibodies having the desired specificity, affinity and/or activity, said clones can be subcloned using serial dilution procedures and cultured using standard techniques; see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), 59-103. It should also be borne in mind that monoclonal antibodies secreted by subclones can be isolated from culture medium, ascites fluid or serum by standard purification procedures such as, for example, protein A chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulation and variable region genes isolated. For example, mononuclear cells can be obtained

- 40 046697 peripheral blood cells from an immunized or naturally immune mammal, such as a human, and cultured for approximately 7 days in vitro. These cultures may be screened for specific IgGs that meet the screening criteria. Cells from wells that show positive results can be isolated. Individual Ig-producing B cells can be isolated using FACS sorting or by identification in a complement-mediated hemolytic plaque assay, Ig-producing B cells can be transferred into the tube using micromanipulation, and the VH and VL genes are amplified using, for example, RT-PCR. The VH and VL genes can be cloned into an antibody-carrying expression vector and transfected into cells (eg, eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to one of ordinary skill in the art. Such techniques are described in various laboratory manuals and major publications. In addition, techniques suitable for use in the present invention as described below are described in: Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) , incorporated herein by reference in its entirety, including appendices.

Antibody fragments that recognize specific epitopes can be prepared using known techniques. For example, Fab and F(ab')2 fragments can be produced recombinantly or by proteolytic digestion of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab') ₂ fragments contain a variable region, a light chain constant region and a heavy chain CH1 domain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving the coupling of immunospecific portions of immunoglobulins with detection reagents such as radioisotopes.

In one embodiment, an antibody of the present invention includes at least one CDR region of an antibody molecule. In another embodiment, an antibody of the present invention includes at least two CDR regions from one or more antibody molecules. In another embodiment, an antibody of the present invention includes at least three CDR regions from one or more antibody molecules. In another embodiment, an antibody of the present invention includes at least four CDR regions from one or more antibody molecules. In another embodiment, an antibody of the present invention includes at least five CDR regions of one or more antibody molecules. In another embodiment, an antibody of the present invention includes at least six CDR regions from one or more antibody molecules. Examples of antibody molecules containing at least one CDR region that may be included in subject antibodies are described herein.

The antibodies of the present invention can be produced by any method known in the art for synthesizing antibodies, in particular by chemical synthesis or, preferably, by recombinant expression techniques as described herein.

In one embodiment, an antibody or antigen binding fragment, variant or derivative thereof of the present invention includes a synthetic constant region in which one or more domains are partially or completely deleted (domain deleted antibodies). In certain embodiments, suitable modified antibodies comprise domain deleted constructs or variants where the CH2 domain has been completely deleted (ΔΟΗ2 constructs). In other embodiments, the deleted domain may be replaced with a short connecting peptide to provide flexibility and freedom of movement of the variable region. Those skilled in the art will appreciate that such designs are particularly preferred due to the regulatory effect of the CH2 domain on the rate of catabolism of the antibody. Domain deleted constructs can be prepared using a vector encoding the human IgG ₁ constant domain, see, for example, WO 02/060955 and WO 02/096948A2. The vector is constructed to remove the CH2 domain and produce a synthetic vector expressing the IgG ₁ constant region with the domains removed.

In certain embodiments, the antibodies or antigen-binding fragments, variants, or derivatives thereof of the present invention are miniantibodies. Miniantibodies can be produced using methods known in the art, see, for example, US patent 5837821 or international application WO 94/09817.

In one embodiment, an antibody or antigen-binding fragment, variant, or derivative thereof of the present invention comprises an immunoglobulin heavy chain containing the deletion or substitution of a minor number of amino acids, or even a single amino acid if this allows binding of monomeric subunits. For example, mutation of a single amino acid in selected regions of the CH2 domain may be sufficient to significantly reduce Fc binding, with

- 41 046697 thus improving localization in the NTT area. Likewise, it may be appropriate to simply remove that portion of one or more constant region domains that controls the effector function (eg, complement fixation) that is desired to be modulated. Such partial deletions of constant regions may improve selected characteristics of the antibody (serum half-life) while maintaining other desired functions associated with the intact constant region domain in question. In addition, as mentioned above, the constant regions of the disclosed antibodies may be synthetic, incorporating mutation or substitution of one or more amino acids to improve the profile of the resulting constructs. It may be possible to disrupt the activity provided by the conserved binding site (eg, Fc binding) while essentially maintaining the configuration and immunogenicity profile of the modified antibody. Other embodiments include the addition of one or more amino acids to the constant region to enhance desired properties, such as effector function, or to provide more efficient attachment of cytotoxins or carbohydrates. In such embodiments, it may be advantageous to insert or replicate specific sequences derived from selected constant region domains.

The present invention also provides antibodies that contain, consist essentially of, or consist of variants (including derivatives) of antibody molecules (e.g., VH regions and/or V _L regions) described herein, wherein said antibodies or fragments thereof are immunospecific contact NTT. To introduce mutations into the nucleotide sequence encoding an antibody, standard techniques known to those skilled in the art may be used, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Preferably, said variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the reference region VH, VH-CDR1, VH-CDR2, VH-CDR3, region V _L , VL-CDR1 , VL-CDR2 or VL-CDR3. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue that has a side chain with a similar charge. Families of amino acid residues with side chains having similar charges have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine). Alternatively, mutations can be introduced randomly throughout the entire coding sequence or part of the coding sequence, for example, by saturation mutagenesis, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity (eg, the ability to bind HTT, and/or mutated and/or aggregated types of HTT, and/or their fragments).

For example, it is possible to introduce mutations only in the framework regions or only in the CDR regions of the antibody molecule. The introduced mutations may be silent or neutral missense mutations, eg, having no or little effect on the ability of the antibody to bind antigen; in fact, some such mutations do not change the amino acid sequence at all. These types of mutations may be suitable for optimizing codon frequency, or improving the production of hybridoma antibodies. Codon-optimized coding regions encoding antibodies of the present invention are described in various sections herein. Alternatively, non-neutral missense mutations may alter the ability of an antibody to bind antigen. Most silent and neutral missense mutations are predicted to be located in framework regions, whereas most non-neutral missense mutations are expected to be located in CDRs, although this is not an absolute requirement. One skilled in the art will be able to design and test mutant molecules that have desired properties, eg, no change in antigen-binding activity or a change in binding activity (eg, increased antigen-binding activity or change in antibody specificity). Following mutagenesis, the encoded protein can be expressed in a conventional manner, and the functional and/or biological activity of the encoded protein (e.g., the ability to immunospecifically bind at least one HTT epitope and/or mutated and/or aggregated HTT species, and/or fragments thereof) can be determined using the techniques described herein or conventional modifications of techniques known in the art.

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III. Antibody-encoding polynucleotides

The polynucleotide encoding an antibody or an antigen binding fragment, variant or derivative thereof may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an antibody or an antigen-binding fragment, variant, or derivative thereof may consist of single-stranded and double-stranded DNA; DNA, which is a mixture of single-stranded and double-stranded regions; single-stranded and double-stranded RNA; and RNA, which is a mixture of single-stranded and double-stranded regions; hybrid molecules containing DNA and DNA, which may be single-stranded or, more commonly, double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide encoding an antibody or an antigen-binding fragment, variant, or derivative thereof may consist of three-stranded regions containing RNA or DNA, or both RNA and DNA. A polynucleotide encoding an antibody or an antigen binding fragment, variant or derivative thereof may also contain one or more modified bases, or DNA or RNA backbones modified for stability or other purposes. Modified bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications to DNA and RNA can be made; accordingly, the polynucleotide includes forms modified by chemical, enzymatic or metabolic means.

An isolated polynucleotide encoding a non-naturally occurring variant of an immunoglobulin-derived polypeptide (eg, a portion of an immunoglobulin heavy chain or a portion of an immunoglobulin light chain) may be produced by introducing one or more nucleotide substitutions, additions, or deletions into the immunoglobulin nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions replace one or more non-essential amino acid residues.

As is well known, RNA can be isolated from parental B cells, hybridoma cells or other transformed cells using standard techniques such as guanidine isothiocyanate extraction and precipitation followed by centrifugation or chromatography. If necessary, mRNA can be isolated from total RNA using standard techniques such as oligocellulose chromatography. Suitable techniques are known in the art. In one embodiment, cDNAs that encode the light and heavy chains of an antibody can be produced, simultaneously or separately, using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by consensus primers corresponding to the constant region, or by specific primers based on published DNA and heavy and light chain amino acid sequences. As described above, PCR can also be used to isolate DNA clones encoding the light and heavy chains of an antibody. In this case, libraries can be screened using consensus primers or larger homologous probes, such as probes corresponding to the human constant region. DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art and, after restriction mapping, sequenced in accordance with standard well-known techniques, described in detail, for example, in the above-mentioned references relating to recombinant techniques. DNA. Of course, the DNA can be represented by synthetic DNA in accordance with the present invention at any time during the extraction process or subsequent analysis.

In this context, the present invention also relates to a polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of an antibody according to the present invention. According to one embodiment, the present invention provides an isolated polynucleotide containing essentially or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein at least one heavy chain variable region CDR region or at least two VH- regions The heavy chain variable region CDRs are at least 80, 85, 90, or 95% identical to the amino acid sequences of the reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 from the antibodies described herein. Alternatively, the _VH -CDR1, _VH -CDR2, or _VH -CDR3 regions of said VH are at least 80, 85, 90, or 95% identical to the amino acid sequences of the reference heavy chain VH-CDR1, VH-CDR2, and VH -CDR3 from antibodies as described herein. Accordingly, in this embodiment, the heavy chain variable region of the present invention comprises VH-CDR1, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in FIG. 1.

According to another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (V _L ), wherein at least one of the regions

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The VL-CDR of said light chain variable region or at least two VL-CDR regions of said light chain variable region are at least 80, 85, 90 or 95% identical to the amino acid sequences of the reference light chain VL-CDR1, VL-CDR2 or VL- CDR3 from antibodies as described herein. Alternatively, the VL-CDR1, VL-CDR2, or VL-CDR3 regions of said V _L are at least 80, 85, 90, or 95% identical to the amino acid sequences of the reference light chain VL-CDR1, VL-CDR2, and VL-CDR3 from the antibodies as described in this document. Accordingly, in this embodiment, the light chain variable region of the present invention comprises VL-CDR1, VL-CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in FIG. 1.

In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein the VH-CDR1, VH-CDR2, and VHCDR3 regions contain polypeptide sequences identical to the groups VH-CDR1, VH-CDR2, and VHCDR3 shown in FIG. 1.

As is known in the art, the sequence identity of two polypeptides or two polynucleotides is determined by comparing the amino acid sequence or nucleic acid sequence of one polypeptide or polynucleotide with the sequence of a second polypeptide or polynucleotide. As described herein, any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 identical to another polypeptide. % or 95%, can be determined using methods and computer programs/software known in the art, for example, but not limited to, using the BESTFIT program (Wisconsin Sequence Analysis Package, version 8 for Unix , Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). The BESTFIT program uses the Smith and Waterman local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489) to find the most homologous segment of two sequences. When using BESTFIT or any other sequence alignment program to determine, for example, 95% identity of a particular sequence to a reference sequence in accordance with the present invention, the parameters are set, of course, to calculate the percent identity over the entire length of the polypeptide reference sequence, and that Gaps in homology are allowed, amounting to up to 5% of the total number of amino acids in the reference sequence.

According to a preferred embodiment of the present invention, said polynucleotide comprises, consists essentially of, or consists of a nucleic acid having the polynucleotide sequence of the VH or VL region of an anti-HTT antibody and/or an antibody recognizing the polyproline region of HTT, and/or mutated and/or aggregated HTT species, and/or their fragments according to the data given in table. II. Additionally, according to one embodiment, said polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a V _H or V _L region polynucleotide sequence of an anti-HTT antibody and/or an antibody recognizing a proline-rich HTT region, and/or mutated and/or aggregated types of NTT, and/or their fragments according to the data given in table. III; and/or also recognizing the C-terminal region of HTT, and/or mutated and/or aggregated types of HTT, and/or their fragments according to the data given in table. IV; and/or also recognizing the Q/P-rich region of HTT, and/or mutated and/or aggregated types of HTT, and/or fragments thereof according to the data given in table. VII. Moreover, according to one embodiment, said polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a VH or VL region polynucleotide sequence of an anti-HTT antibody and/or an antibody that recognizes HTT, and/or mutated and/or aggregated HTT species, and /or their fragments according to the data given in table. V. In addition, according to one embodiment, said polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a V _H or V _L region polynucleotide sequence of an anti-HTT antibody and/or an antibody recognizing the N-terminal region of HTT, and/or mutated and/or aggregated types of NTT, and/or their fragments according to the data given in table. VI. Additionally, according to one embodiment, said polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a V _H or V _L region polynucleotide sequence of an anti-HTT antibody and/or an antibody recognizing HTT, and/or mutated and/or aggregated HTT species , and/or their fragments according to the data given in table. V. According to an additional or alternative embodiment, the specified polynucleotide contains, consists essentially of or consists of a nucleic acid having the polynucleotide sequence of the VH or VL region of an anti-HTT antibody and/or an antibody corresponding to those shown in table. VIII.

It will be apparent to one skilled in the art that polynucleotides encoding at least a light and/or heavy chain variable domain may encode the variable domain of both immunoglobulin chains or just one chain. According to one embodiment, respectively, said polynucleotide contains, consists essentially of, or consists of a nucleic acid having the sequence of polynucleotides of the VH and VL region of an antibody to HTT and/or fragments thereof according to the data given in table. II, III, IV, V, VI, VII or VIII.

Table II. The nucleotide sequences of the VH and VL region of antibodies recognizing the epitope of the polyproline region of HTT, i.e. aggregated form of the protein encoded by exon 1

Antibody Nucleotide sequences of the variable region of the heavy ( _VH ) and variable region of the light ( _VL ) chains NI-302.33C11-V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGG G AACTCCCTG AG ACTCTCCTGTG CAG CGTCTG G ATTCAG GTTCAGT GACTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTG GAGTGGCTGGCACTTATATGGTATGATGGAGGGTATAAGTACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAA GAATACGATGTTTCTACAAATGAACAGCCTGAGAGCCGAGGACACG GCTGTTTATTACTGTGCGACCCACCTAGAATATTGCAGTAGAACCAC CTGCTATCTCGGCCACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCG SEQ ID NO: 1 NI-302.33C11-V _k GACATCCAGTTGACCCAGTCTCCGTCCTTCCTATCTGCGTCTGTGG GAGACACAGTCACCTTCACTTGCCGGGCCAGTCAGGGCATTAGCG ATTATTTAGCCTGGTTTCAGCAGAAACCAGGGATTGCCCCTAAGCT CCTGATCTATGCTGCGTCCACTTTGCAAACCGGGGTCCCATCAAGG TTC AG CG G CAG TGGATCTGG G AC AG AATTC ACTCTC AC AATCC G C A GCCTG CAGTCTG AAG ATTTTG G AACTTATTACTGTCAG CAG CTTAAA ACTTACCCGTACACTTTTGGCCAGGGGACCAAGGTGGAAATCAAA SEQ ID NO: 3 NI-302.74C11-V _h GAGGTGCAGCTGGTGCAGTCTGGGACTGAGGTGCAGAAGCCTGGG GCCTCAGTAAAAGTCTCCTGCAAGGCTTCTGGATACAGTTTCACCG GCTACTTTTTGCACTGGGTACGACAGGCCCCTGGACAAGGGCTTGA GTGG ATG G GGTG G ATCAACCCTAAC AGTG GTG ACACAAACTATG CA GAGAAGTTTCGGGGCAGAATCATCATGACCAGGGACACGTCTGTCA GCACAGCCCACATGGAGTTGAGCAGCCTGAGATTTGACGACACGG CCCTATATTACTGTACGAGAGAGGCCCCTGACCCGGGCGCTGAGA CGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 25 NI-302.74C11-V _l CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGA CAGACGGCCAGGATCACCTGCTCTGGAGATGCAGTGCCAAAGCAG TATATTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTATTCTGG TGATATATAAAGACACTCAGAGGCCTTCAGGGATCCCTGAGCGATT CTCTGGCTCCAACTCAGGGACAACAGTCACGTTGACCATAACTGGC

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GTCCAGGCAGACGACGAGGGTGACTATTACTGTCAATCAGCAGACA GTAGTGCTACTTGGGTGTTCGGCGGAGGGACCAAATTGACCGTCCT A SEQ ID NO: 27 NI-302.15F9-V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCACGCCGGG GGGGTCCCTGAGACTCTCGTGTGAGGCCTCTGGATTTCTCTTCAAG AATTCTAGCATGAACTGGGTCCGTCAGACTCCGGGGAAGGGGCTG GAGTGGGTCTCGTCCATTGACACTTCTGCTACAAATTATAAGTATTA TGCAGACTCTGTGAAGGGCCGATTTACCATCTCCAGGGATGACGCC ACCAACTCTCTCT ATCTGCAAATGAATAGCCTGCGAGCCGACGACA CGGCTACTTATTACTGTGCGCGAGGTTATTATACCCCCCGGGACTT TGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 29 NI-302.15F9- V _K GATGTTGTGATGACTCAGTCTCCACAGACCCTGTCCGTCAGCCTTG G ACAG G CG G CCTCCATCTCCTG CAG GTCG AGTCAAAGCCTCTTGTA TCGTGATAACAACACATACTTGAATTGGTTTCACCAGAGGCCAGGC CAATCTCCAAGGCGCCTCATTTATAGGGCTTCTGACCGGGACTCTG GGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCA CATTGA AAATCAGTGGAGTGGAGGCTGAAGATGTTGGCACTTATTA CTG CATG C AAG G AACACACTG GCCTCG G ACGTTCG GCC AAG GG AC CAAG GTG G AG ATCAAA SEQ ID NO: 31 NI-302.39G12-V _H GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCACCCTTGG GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTA ATTACGCCATAACTTGGGTCCGCCGGGCTCCAGGGAAGGGGCTGC AATATATTTCAG TAATTTATCGTG ATG G CAG G ACATACTACG G AG AC TCCGTGAGGGGCCGCTTCACCATCTCTAGGGACGATTCCAAGAACA CTCTCTATCTTCAAATGAACAGCCTGAGATTTGAGGACACGGCTGT GTATTACTGTGCGAGAGCGCACGGCCAATATTACTATGGTGTGGAC GTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 33 NI-302.39G12-V _K GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCAGCCCTG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACA TAGTAATGGATACAACTATTTGGATTGGTACCGGCAGAAACCAGGG CAGTCTCCACAGCTCCTGATCTATTTGAGTTCTAATCGGCCCTCCG GGGTCCCTGATAGGTTCAGTGCCAGTGGATCAGGCACAGAGTTCA CACTGC AAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTA CTG CATG C AATCCTG CAAACGTTCACTTTCG G CG GAGG G ACCAAA GTGGATATCAAA SEQ ID NO: 35 NI-302.11A4- V _H GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCGGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCCCCGTCAG TAGCAGTTACATGAGCTGGGTCCGCCAGGCTCCAGGAGAGGGGCT GGAGTGGGTCTCAGTTCTTTATAGAGACGGTGACACATACTACGCA GACTCCGTGCAGGGCCGATTCACCATCTCCAGAGACAATTCCCAGA ACACGTT CTATCTTCAAATGAACAGCCTGAAAGCCGAGGACACGGC CGTGTATTACTGTGCGGGTGATAGAAGGTCGTCACACTACTATTAC GGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 37 NI-302.11A4-V _K GAAATTGTGATGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAG GAGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCA GCAGCTACTTCGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATGGTACGTCCCGCAGGGCCACTGCCATCCCAGAC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCA GCAGACTGGAG CCTGAAGATTTGCAGTGTATTACTGTCAACAGTAT GGTAGCTCGTGGACGTTCGGCCCAGGGACCAAGGTGGAGATCAAA

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SEQ ID NO: 39 NI-302.22H9- V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCACCCTTGG GGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCTA ATTACGCCATAACTTGGGTCCGCCAGGCTCCAGGAAGGGGCTGG AATATATTTCAGTGATTTATCGTGATGGCAGGACATACTACGGAGAC TCCGTGAGGGGCCGCTTCACCATCTCTAGGGACGATTCCAAGAACA CTATCTAT CTTCAAATGAACAGCCTGAGATTTGAGGACACGGCTGT GTATTACTGTGCGAGAGCGCACGGCCAATATTATTATGGTGTGGAC GTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO 41: NI-302.22H9- V _K CTG AC AATC AG CAG AG TG G AG G CTG AG G ATG TTG G G G TTTATTACT GCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCAAGGT GGAGATCAAA SEQ ID NO: 43 NI-302.44D7- V _H GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC AGCTATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTCTCAGGTATTGGTTATAGTGATACTAGCACATATTACGC AGACTCCGTGAAGGGCCGCTTCACCGTCTCCAGAGACATTTCCAAG GCTGTATCTGCAATGAATAGCCTGAGGGCCGAGGACACG GCCGTATATTACTGCGCGAAAGGTACCAGGGACTATTACGGTATGG ACGTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCG SEQ ID NO: 45 NI-302.44D7- V _K CAGACTGTGGTGACTCAGGAGCCATCGTTCTCAGTGTCCCCTGGAG GGACAGTCACACTCACTTGTGGCTTGAGTTCTGGCTCAGTTTCTACT AGTTACTACCCCAGCTGGTACCAGCAGACCCCAGCCGGGCTCCA CGCACGCTCATCTACAGCACAAACACTCGCTCTTCTGGGTCCCTG ATCGCTTCTCTGGCTCCATCCTTGGGAACAAGGCTGCCCTCACCAT CACGGGGG CCCAGGCAGATGATGAATCTGATTATTACTGTGTGCTG TTTATGGGTAGTGGCATTGGGGTGTTCGGCGGAGGGACCAGGCTG ACCGTCCTA SEQ ID NO: 47 NI-302.37C12-V _H GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTGGTCCAGCCTGGG GGGTCCCTGAGACTCTCTTGTGTTGCCTCTGCACTCACCGTCACTA ACAGCCAAATGACCTGGGTCCGCCGGGCTCCAGGAGGGGGTTG GAGTGGGTCTCAGTTATTTTACACCAGTGGTAGTGCATACTACGCAG ACTCCGTGAAGGGCAGATTCACCATCTCCAGGACAATTCCAAGAA CACAGTG TTTCTTCAAATGAACAGCCTGAGAGTCGAAGACACGGCT GTGTATTACTGTGCGAAAGGCCCATCAGCCTATTATTACGGTTTGGA CCTTTGGGGCCAAGGGACCACGGTCACCGTCTCCTCG SEQ ID NO: 49 NI-302.37C12-V _k GATATTGTGATGACTCAATCACCACTCTCCCTGCCCGTCACCCCTG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGC ATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCGGG GCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTA CACTGAAG ATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTA CTG CATG C AAG GTCTACAG ACGTACACTTTTG GCCAG GG G ACCAAG CTGGAGATCAAA SEQ ID NO: 51

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NI-302.55D8- V _H CAGGTGCAGCTGGTGCAGTCTGGGTCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCG ACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGA GTGG ATG G G ACG G ATCAACCCTAAC AATG GTGG CACAAACTATG CA CAG AACTTTCAGG G CTG G GTCACCATG ACCAG GG ACACGTCCAT CA GCCAAGCCTACATGGAGCTCAGCAGACTGAGATCTGACGACACGG CCGTCTATTACTGTGCGAGAGTGGGGGGCGAGCTGCTACGAGAAG GCGGCTATCACTACTACATGGACGTCTGGGGCAAGGGGACCACGG TCACCGTCTCCTCG SEQ ID NO: 53 NI-302.55D8- V _L CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGG CAG AG G G TCACCATCTCCCTGCACTG GG AAC AG CTCCAACATCG GG GCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCC CCAAACTCCTCATCTTTGATAATACCAATCGGCCCTCAGGGGTCCC TGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGG CC ATCACTGG G CTCCAG GCTG AG G ATG AG G CTAATTATCACTG CCAGT CCTATGACAACAGCCTGAGTGGTTCTTGGGTGTTCGGCGGAGGGA CCAAGCTGACCGTCCTA SEQ ID NO: 55 N1-302.7A8- V _H A ACTCACTGTATCTG CAG ATG AACAG CCTG AG AGCCG AG G ACAC GGCTGTATATTACTGTGCGAGAGGAGATTATAATTCGGGCATCTATT ACTTTCCCG GG G ACTACTG GG G CCAG GG CACCCTG GTCACCGTCT CCTCG SEQ ID NO: 57 N1-302.7A8- V _K A CAGCAGGGTGGAGGCTGAGGATGTTGGCATTTATTA CTGCATGCAAGGTACACACTGGCCTGGGACGTTCGGCCAAGGGAC CAAG GTG G AG ATCAAA SEQ ID NO: 59 NI-302.78H12-V _h CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCG GAGACCCTGTCCCTCACCTGTCTTGTCTCTAGTTACTCCATCAGCAA TGGTTACTACTGGGGCTGGATTCGGCAGCCCCCAGGGAAGGGGCT GGAGTGGATTGGGAGTATCTATCATAATGGGAACACCTATTACAAC CCGTCCCTCAAGAGTCGAGTCATCATTTCAGTAGACACGTCCAAGA ACCAGTTCT CCCTGAAGTTGAGGTCTGTGACCGCCGCAGACACGG CCGTGTACTACTGTGCGATGCCAAGTGCCACCTATTATTATGGTTC GGGGACTCAATTCCATGCGTTTGATGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCG SEQ ID NO: 61 NI-302.78H12-V _l G CTCCAG G CTG AG G ATG AG G CTG ACTATTACTG CTG CTC ATATGCTGGCAGTTACACCTTCGAGGTATTTGGCGGAGGGACCAAG CTGACCGTCCTA

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SEQ ID NO: 63 NI-302.71F6- V _H CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTATTGAAGCCTTCG GAGACCCTGTCCCTCACGTGCGCTGTCTATGGTGGGTCCCTCAGT GGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTG GAGTGGATAGGGGAAGTCAATCATAGTGGAGGCACCAACCTCAATT CGTCCCTCAAGAGTCGAGTCATCATTTCAGTAGACAAGTCCAAGAA GCAG TTCTCCCTGAAACTGAGCTCTGTGACCGCCGCGGACACGGC TATGTACTTCTGTGCGAGAGGATACAGCTATGACCCAAAATACTACT TTGACTCCTGGAGCCAGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 65 NI-302.71F6-V _l CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGAC AGGCGATCACCATCTCCCTGCACTGGAACCAGTAGTGATATTGGGAG TTATGATTTTGTCTCCTGGTACCAGCAGGACCCAGGCAAAGCCCCC AAAGTCATTATTTATGGGTCAATAAGCGGCCCTCAGGGGTTTCTAA TCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATC TCT GGACTCCAGGCTGACGACGAGGCTGATTATTACTGCTGCTCAT ATGCTGGTAGTACCACTTGGGTGTTCGGCGGAGGGACCAAACTGA CCGTCCTA SEQ ID NO: 67 NI-302.11H6-V _h A CCACAGACACATCCGCG AGTACAGCGTACATGGAACTGAGGAGCCTCAGATCTGACGACACG GCCATCTATTATTGTGCGGCTGTAGACACCACTTACTACTATTACGG CATGGACGTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 69 NI-302.11H6-V _l CAGACTGTGGTGACTCAGGAGCCAACGTTCTCAGTGTCCCCTGGA GGGACAGTCACACTCACTTGTGCCTTGAGGTTTGGCTCAGTCTCTA GTAG CTACTATC CC AG CTG G TTCC AG CAG ACC CC AG GCCAGGCTC CACGCACGCTCATCTACAGCACAAACACCCGCTCTTCGGGGGTCC CTG CTCG ATTCTCTG G CTCCATTCTTG G G AACAAAG CT GCCCTCAC CATCGCGGGGGCCCAGGCAAATGATGAGGCTGACTATTACTGTGT GCTGTATATGGGTAGTGGAATCGGGGTGTTCGGCGGAGGGACCAA GTTGACCGTCCTA SEQ ID NO: 71 NI-302.3D8- V _H GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTCTCTTGTGAAGCCTCCGGATTCATCTTTAAA ACCTATGCCATGAGCTGGGTCCGCCAGCTTCCCGGGAGGGGGCTG GAATGGGTCTCAGCTATAAGTGCCACTGGTGGAAGCACCTTCTACG CAG AG TCCGTG AAG G GCCG GCTC ACCATTTCCAG AG ACACTG CCA AGA ATACAGTGTATCTGCAAATGAACAACCTGAGAGCCGAAGACAC GGCCATGTATTACTGTGCGAAAGGGTCGACTGCGGTATATCTCTTT GACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 73 NI-302.3D8- V _K GACATCCAGATGACCCAGTCTCCGTCCTCACTGTCTGCATCTGTAG GGGACAGAGTCACCCTCACTTGTCGGGCGAGTCAGGACATCAGAA ATTTCTTGGCCTGGATTCAGCAGAAGCCAGGGAAACCCCTAAGTC CCTGATCTATGCTGCGTCCACTTTGCAAAGTGGGGTCCCATCACGA TTCAG CG G CAGTG G ATCCGGG ACAG ATTTC ACTCTCACC ATCAG CA GCCTGCACCCTGAAGATTTTGCTACTTATTACTGCCAGCAGTTTTAT AATTACCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 75 NI-302.18A1-Vh CAG CTG CAG CTG CAG G AGTCG GG CCCAGG ACTAGTG AAG CCTTCG GAGGCCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCACTA

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CTGATTATTACTATTGGGGCTGGATCCGCCAGTCCCCAGGCAAGGG ACTAGAGTGGGTTGGGACAATATTTGGTGGGGCCACCTACTAC AATCCGTCCCTCAGGAACCGGGTCTCGATATCTGTGGACACGTCCA ACACTCGCCTCTCCCTGAGACTTATCTCTCTGAGCGCCGCTGACAC GGCCGTCTATTATTGTGCGAGAGTGGGCTACTTGGATAGGAGTGGT CTTCTTGTGGGCC AGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 77 NI-302.18A1-V _K GAAATTGTGCTGACGCAGTCTCCACTCTCCGTGCCCGTCACCCCCG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGC ATAATAATG G ATACAACTATTTG G ATTG GTACCTG AAG AAG CCTG G G CAGTCTCCACAACTCCTGATCTATTTGGGCTCTACTCGGGCCTCCG GGGTCCCTGACAGGTTCAGTGCCAGTGGATCAGGCACAGACTTTAC ACTGGAAATCAGCAGAGTGGAGGCTGAAGATGTTGGCGTTTACTAC TGCATGCAAGCTCTGCAGACTCCTCCGACTTTCGGCAGAGGGACCA AGGTGGAGATCAAA SEQ ID NO: 79 NI-302.52C9- V _H GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAACCTGGG GGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCGTCAGTG ACACCTACATGAGTTGGGTCCGCCAGGCTCCAGGAAGGGGCTGG AGTGGGTCTCAGGTATTCATGCCGGTGGTGAAACATATTACGCAGA CTCCGTGAAGGGCCGATTCACCATCTCCAGGACAACTCCAAGAAC ACGCTGTA TCTTCAAATGAATAGGCTGACACCTGAGGACACGGCTG TCTTTTATTGTGCGAGACACTACTACGGTAATGACGACGACACTGAT TATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 85 NI-302.52C9- V _K GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGC ATAGTAATGGATACAACTATTTGGATTGGTACGTGCAGAAGCCAGG GCAG TCTCC ACAG CTCCTC ATCTATTTG G GTTCTACTCGG G CCTCC GGGGTCCCTGACAGATTCAGTGGCAGTGGATCAGGCACAGATTT TA CACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTA CTGCTTACAAGCTCAACAAATTCCGTGGACGTTCGGCCAAGGGACC AAG GTG G AG ATCAAA SEQ ID NO: 87 NI-302.46C9- V _H CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTAAAGCCTTCA CAGACCCTGTCCCTCACCTGCACTGTTTCTGGTGCCTCCGTCAGCA GTGGTGCCTACTACTGGAGTTGGATCCGGCAGCCCGCCGGGAAGC GACTGGAGTGGATTGGGCGTGTCTATCCCACTTGGAGCACCAACTA CAACCCCTCCCTCGAGTCGAGTCACCATATCGTTAGACACGTCC AACAACCAGTTCT CCCTGAAGCTGACCTCTTTGACTGCCGCAGACA CGGCCGTTTATTACTGTGCGAGAGAGGCTCCTGGTGACTACGATGC TGCGCCCCTAGCCTACTGGGGCCAGGGCACCCTGGTCACCGTCTC CTCG SEQ ID NO: 89 NI-302.46C9- V _K GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTG GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGTACATTAGCCA CTATTTAAATTGGTATCGGCAGAAACCAGGGAAAGCCCCTCAGCTC GTAATCTATGCTGCATCCAGTTTGCAAAGTGAGGTCCCATCAAGGTT CAGTGGGAGTGGATCTGGGCCAGAGTTCACTCTCACCATCAGCAGT CTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACAC TACCCCTCG AACTTTTG GCCAG G GG ACCAAG CTG GAG ATCAAA SEQ ID NO: 91

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Table III. The nucleotide sequences of the VH and VL region of antibodies recognizing the epitope of the proline-rich region of HTT, i.e. aggregated form of the protein encoded by exon 1

Antibody Nucleotide sequences of the variable region of the heavy ( _VH ) and variable region of the light ( _VL ) chains NI-302.63F3- V _H CAGGTGCAGCTGGTGCAATCTGGGTCTGCGTTCAAGAAGCCTGGG ACCTCAGTGAAAGTTTCCTGCAAGGCCTCTGGATACACCTTCGAGA CCCGTTCTATG AACTG G GTG CG ACAG G CCCCTG G ACAAG GG CTTG AATACATGGGATGGATCAACACCAACACTGGCAACCGCACGTATGT CCAGGCCTTCAGAGGACGATTTGTCTTCTCCTTGGACACCTCTGTC AG CACG GCATATCTG CAG ATCAG CAACTTAAAG ACTG AGG AC ACTG CCGTGTATTACTGTGCGAGAGGGGCAGGTGGGGGATATTGGTTCG ACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 5 NI-302.63F3-V _K GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCAATCAGAGTCTTTTTCTA CAGTTCCAACAATAAC AACTACTTAG CTTG GTAC CAG CACAAATCCG GACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATC CGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTT CTCACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATT ACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACC AAGCTGGAGATCAAA SEQ ID NO: 7 NI-302.31F11-V _H GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCTTGATCCAGCCGGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAG CAGCACCTACATGAGTTGGGTCCGCCAGGCTCCAGGAAGGGGCT TGAGTGCGTCTCAGTTATTTTTAGTGGCGCTGACACATATTACGCAG ACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAA CACACTG TTTCTTCAGATGAACAGCCTGAGAGTCGAGGACACGGCC ACATATTACTGTGTGAGACATTATTATGGTTCAGACCTTCCATCTGA CTTCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 13 NI-302.31F11-V _k GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCGCCCCTG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTATA CAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGG AAGCCTCCACAGCTCCTGGTCTATTTGGGTTCTGATCGGGCCTCCG GGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCAAAGATTTTAC ACTGAA CATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTAC TGCATGCAAGGTCTACAAAGTCCGTGGACGTTCGGCCAAGGGACC AAGCTGGAGATCAAA SEQ ID NO: 15 NI-302.2A2- V _H A ACTCACTGTATCTG CAAATG AACAG CCTG AG AG ACG AG G AC ACG GCTGTGTATTACTGTGCGAGAGGGGACGGCAGTGGCTGGAACGTC TACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 17 NI-302.2A2- V _K GACATCCAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTCTTTTATA CACCTCCAAAAATAAGGACAGTAAGAACTACTTAGGTTGGTACCAG CAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTA CCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTG GGACAGATTTCACT CTCACCATCAGCAGCCTGCAGGCTGAGGATGT

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GGCAGTTTATTACTGTCAGCAGTATTATACTACTCCTCAGTTCGGCG GAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 19 NI-302.15D3-V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTTCAGCCTGGG GG GTCCCTAAG ACTCTCCTGTG CAG CCTCTG G ATTCACCTTCAGTA GCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGTCTGG TGTGGGTCTCACGTATTAGTAATGATGGCAGTAGCAAAACCTACGC GGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAA CACG CTGTATCTG CAAATG AAC AGTCTG AG AGCCG AG G AC ACG GCTGTGTATTACTGTGCAATACTTGGCGGATATTGTAGTAGTACCAG TTGTCGTCCCTTTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCG SEQ ID NO: 135 NI-302.15D3-V _L CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGAC AGTCGATCACCATCTCCCTGCACTGGAACCAGCAGTGACGTTGGTGT TTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCC AAACTCATGATTTTTGATGTCAGTAATCGGCCCTCAGGGATTTCTAA TCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATC GCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCAT ATACAAGCAGCGACACTTGGGTGTTCGGCGGAGGGACCAAGCTGA CCATCCTA SEQ ID NO: 137 NI-302.64E5- V _H GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTAAAGCCTGG GGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGAC CAGGCCTGGATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCT GGAGTGGGTTGGCCGGATTAAAACGAAAACTGAGGGTGAAGCAAC AGACTACGCAGCGCCCGTGAGAGGCAGATTCACCATCTCAAGAGAT GATTCAGAAGAC ACGGTGTTTCTGCAAATGAACAGCCTGAAAACCG AGGACACAGCCCTGTATTACTGTACGTCAACGGGAGTCTTAGCAGC AGCTGTCGATGTCTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCG SEQ ID NO: 164 NI-302.64E5- V _K GACATCCAGTTGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATGACCTGCAAGTCCAGCCAGAGTCTTTTTCTA CAGTTACAACAATGAGAACTACTTAGCCTGGTATCAGCAGAGACCA GGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAAT CCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATT TCACTCTCA CCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTA TTACTG TCAG CAATATTATAGTACTCCTC AG ACGTTCG GCCAAG G GA CCAAAGTGGATATCAAA SEQ ID NO: 168

Table IV. Nucleotide sequences of the VH and VL region of antibodies recognizing the epitope

The C-terminal region of HTT, i.e. aggregated form of the protein encoded by exon 1

Antibody Nucleotide sequences of the variable region of the heavy ( _VH ) and variable region of the light ( _VL ) chains NI-302.35C1-Vh A GTACTTGCAAATGAACGGCCTGAGAGCCGAGGACAC GGCCATCTATTACTGTGTGAAAGGAATTGCCTCCGATAGTAGTGGT TATTCTGCCTTCTGGGGCCCGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 9 NI-302.35C1-Vk GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAAAGTGTTGACA ACCAGTTTGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCT CCTCATTTATGATGCATCCAGGAGGGCCCCTGGCATCCCAGACAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATTAGCA GCCTAGAGCCTGA AGATTTTCGCAATTTATTACTGTCAGCATCGTTAC ACCTGGCTCTACACTTTTGGCCAGGGGGACACGACTGGAGATTAAA SEQ ID NO: 11 NI-302.72F10- V _H GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTCGTACAGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGC AGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTG GAGTGGGTGTCAGATATCAGTGGTATTGGTAGTAACACATACTACG CAGACTCCGTGAAGGGCCGTTTCACCATTTCCAGAGACAATTCCGA CAATACGTTG TACCTGGACATGAGCAGCCTGAGAGCCGAGGACAC GGCCAGATATTACTGTGCGAAGGATCGAAAGCGCAGTGGCTGGTA CGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 176 NI-302.72F10- V _K GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTACAGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGC AGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTG GAGTGGGTGTCAGATATCAGTGGTATTGGTAGTAACACATACTACG CAGACTCCGTGAAGGGCCGTTTCACCATTTCCAGAGACAATTCCGA CAATACGTTG TACCTGGACATGAGCAGCCTGAGAGCCGAGGACAC GGCCAGATATTACTGTGCGAAGGATCGAAAGCGCAGTGGCTGGTA CGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 178

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Table V. Nucleotide sequences of the VH and VL region of antibodies recognizing HTT species and/or their fragments

Antibody Nucleotide sequences of the variable region of the heavy ( _VH ) and variable region of the light ( _VL ) chains NI-302.6N9- V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTGCAGCCTGG GGGGTCCCTGAGACTCTCCTGTGTAGTCTCTGGATTCACCTTTAGT AGTTATGCCATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GCCTGGGTCTCAACAATTAGTGCTACTGGTGGTAGTACATTCTACA CAGACTCCGTGAGGGGCCGGTTCACCATCTCCCGAGACAATTCCAA ACACTGTATCTGCAATGAATAGCCTGAGAACCGACGACACG GCCATATATTATTGTGTGAAAGATCTATTTGGAGTGGACACCTCCTA CTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCG SEQ ID NO: 21 NI-302.6N9- V _K GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGCCCAGTCAGAGTGTCAGCG GCAGGTATGTGGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCA GG CTCCTCTTCTATG CTG CATCCAACAG G G CCATTG GCATCCCAG A CAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGC AGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACT ATGGTGCCTCATCGTACACTTTTGGCCCGGGGGACCAAAGTGGATAT CAAA SEQ ID NO: 23 NI-302.8F1-V _h GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGAAGCCGGG GGGGTCCCTTACAATCTCCTGTGCAGCCTCTGGTTTCACCTTCAGT AATGCCTGGATGAACTGGGTCCGCCAGGCTCCAGGTAAGGGGCTG GAGTGGGTCGGCCATATTAGAACGCAAGCTGAAGGAGGGACATCA

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GACTATGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATG ACTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGA GGACACAGCCGTATATTATTGTATCCCCCCCCCCTACTACTATT ACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCT CG SEQ ID NO: 81 NI-302.8F1-V _L CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGAC AGTCGATCACCATCTCCCTGCACTGGAGCCAGCAGTGATGTTGGGAC TTATGACCTTGTCTCCTGGTACCAACAACATCCAGGCAAAGCCCCC AAACTCATTATTTATGAGGTCAATAAGCGGCCCTCAGGTTTCTTA TCGCTTCTCTGCCTCCAAGTCTGCCAACACGGCCTCCCTGACAATA GCTCCAGGCTGAGGACGAGGCTGAATATTACTGCTGCTCAT ATGCAGGTTATAGCACGGTATTCGGCGGAGGGACCAAGCTGACCG TCCTA SEQ ID NO: 83 NI-302.4A6- V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC GCTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG G AGTGG GTCTCAACTATTAGTG GTAGTG GTG GTAG TACATACTACG CAGACTCCGTGAAGGGCCGGTTCTCCATCTCAGAGACAACTCCAA AAA CACCCTGTATCTG CAAATG AACAG CCTG AG AG COG AG G ACACG GCCGTATATTTCTGTGCGAAAGTTACCACGGAACTCTACGGTGCTA ACTCCTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGT CACCGTCTCCTCG SEQ ID NO: 184 NI-302.4A6- V _K GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGTCA GCAGGTATTTAGCCTGGTACCAGCAAAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATG GTG CATCCAG CAGG G CC ACTG GCATCCCAG AC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCA GC AGACTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAGCTGTAT GGTAACTCACAGACGTTCGGGCCAGGGGACCAAGGTGGAGATCAAA SEQ ID NO: 186 NI-302.12H2-V _h GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTTTCCTGTGAAGCCTCTGGATTCACCTTTAGC AACTATG CCATGG G CTG GG TCCG CCAG G CTCC AG GG AAGG G G CTG GAGTGGGTCTCAGTAATTAGTGGTACTGGTGGTAGCACATACTACG CAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAT GAACACGCTGTATCTGCAAATGAACAGCCCGAGAGCCGACGACAC GGCCGTATATTACTGTGCGAAAGATCTGAGGAAGATTAGCGGTCCT TTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCCTCG SEQ ID NO: 188 NI-302.12H2- V _K GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTTTCCTGTGAAGCCTCTGGATTCACCTTTAGC AACTATG CCATGG G CTG GG TCCG CCAG G CTCC AG GG AAGG G G CTG GAGTGGGTCTCAGTAATTAGTGGTACTGGTGGTAGCACATACTACG CAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAT GAACACGCTGTATCTGCAAATGAACAGCCCGAGAGCCGACGACAC GGCCGTATATTACTGTGCGAAAGATCTGAGGAAGATTAGCGGTCCT TTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCCTCG SEQ ID NO: 192 NI-302.8M1-V _h GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAAGTTTCCTGCAAGGCATCCGGATACACCTTCACCA TCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGA GTGGATGGGAGGAATCAGCCCGAGTGGTGCCCACACAATGTACGC ACAGAATTTCCAGGGCAGAGTCACCGTGACCAGGGACACGTCCAC GAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACAC GGCCGTGTATTACTGTGCGAGAGGGAGCACGGTGACTAACTATCG ACCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC G SEQ ID NO: 194 NI-302.8M1-V _k GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG GAGACAGAGTCACTATCACTTGCCGGGCGAGTCAGGACATTAGCAA TTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAACTCC TGATCTTTGCTGCATCCACTTTGCAATCAGGGGTCCCGTCTCGGTT CGGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGC CTG CCTG AAG ATGTTG CAACTTATTACTGTCAAAACTATAACAG TGGCCCTCCGCCTTTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO: 198

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Table VI. The nucleotide sequences of the VH and VL region of antibodies recognizing the epitope of the N-terminal region of HTT, i.e. aggregated form of the protein encoded by exon 1

Antibody Nucleotide sequences of the variable heavy (Vh) and variable light ( _VL ) chains NI-302.15E8-V _H A ACTC ACTCTATCTG CAAATG AAC AG CCTG AG AG ACG AGG AC ACGGCTGTGTATTACTGTGCGAGAGCAGGGGACTTCGGGGAGTTA CTCACTGGTGAGGGGTATTACGGTATGGACGTCTGGGGCCAAGGG ACCACGGTCACCGTCTCCTCG SEQ ID NO: 131 NI-302.15E8- V _L TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGAC AG ACAG CCASS ATCACCTG CTCG GG AG ATG AATTGG G G GATAAATA TGTTGGTTGGTATCAACAGAAGCCAGGCCAGTCCCCTCTGCTGGTC ATCTATCAAGATGCGAAGCGGCCCTCAGGGATCCCTGAGCGATTCT CTG GCTCCAACTCTG G G AACAC AG CCACTCTG ACC ATCAG CGG G A CCCAGGCTATGGATGAGGCTGACTACTACTGTCAGGCGTGGGACA GCGGCACGATGGTTTTCGGCGGAGGGACCAGGCTGACCGTCCTA SEQ ID NO: 133

Table VII. The nucleotide sequences of the V _H and V _L region of antibodies recognizing the epitope of the Q/P-rich region of HTT, i.e. aggregated form of the protein encoded by exon 1 Antibody Nucleotide sequences of the variable region of the heavy (Vh) and variable region of the light ( _VL ) chains

N1-302.7D8-V _H CAGGTGCAGCTGGTGCAATCTGGATCTGAGTTGAAGAAGCCTGGG

GCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACAACTTCAATAA CTATG SCATS AATTG GTTG CG ACAG G CCCCTG G ACAAG GG CTTG AG TGGATGGGATGGATCAACACCATCACTGGGCACCCAACGTATGCCC AGGGCTTCAAAGGACGATTTGTCTTCTCCTTGGACACCTCTGTCAG

CACGGCATATCTGCAGATCAGCAGCCTAAAGCCTGAGGACACTGCC GTCTATTACTGTGCGAGAACTTACAGTAACTACGGCGAATTTGACTA CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO: 172 NI-302.7D8- V _L CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCGTGGA CAGTCG ATCACCATCTC CTG CACTG G AACCAG CAGTG ATGTTG G AA GTTATAACCTTGTCTCCTGGTACCAACAGTACCCAGGCAAGGCCCC CAAGCTCATAATTCATGAGGGCAGTGAGCGGCCCTCAGGGGTTTCT AATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTG ACAA TTTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTC ATATGCAGGTACTACTACTTTCGTGCTATTCGGCGGAGGGACCAAG CTGACCGTCCTC SEQ ID NO: 174

Due to the cloning strategy used, the amino acid sequence at the N-terminus and C-terminus of the heavy and light chains could potentially include primer-induced changes in the FR1 and FR4 regions without substantially affecting the biological activity of the antibody. To obtain a human consensus antibody, the nucleotide and amino acid sequences of the original clone can be aligned and rearranged according to the corresponding human germline variable region sequences from the database; see for example Vbase2 as described above. Human antibody amino acid sequences are shown in bold when it is believed that the N- and C-terminal amino acids may potentially differ from the germline consensus sequence as a result of the action of the PCR primer and are accordingly replaced using primer-induced mutation correction (PIMC) ), see table. VI. Accordingly, according to one embodiment of the present invention, said polynucleotide contains, consists essentially of, or consists of a nucleic acid having the sequence of polynucleotides of the VH and VL region of an antibody to HTT and/or fragments thereof, as shown in Table 1. VI.

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Table VIII. Nucleotide sequences of the VH and VL region of antibodies recognizing HTT types, and/or their fragments; substitutions obtained using PIMC are highlighted (in bold)

Alternative antibody regions generated using PIMC Nucleotide sequences of the variable region of the heavy ( _VH ) and variable region of the light ( _VL ) chains NI-302.33C11PIMCVh CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGG GAACTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCAGGTTCAGT GACTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTG GAGTGGCTGGCACTTATATGGTATGATGGAGGGTATAAGTACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAA GTTTCTACAAATGAACAGCCTGAGAGCCGAGGACACG GCTGTTTATTACTGTGCGACCCACCTAGAATATTGCAGTAGAACCAC CTGCTATCTCGGCCACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCG SEQ ID NO: 97 NI-302.33C11PIMCVk GACATCCAGTTGACCCAGTCTCCGTCCTTCCTATCTGCGTCTGTGG GAGACACAGTCACCTTCACTTGCCGGGCCAGTCAGGGCATTAGCG ATTATTTAGCCTGGTTTCAGCAGAAACCAGGGATTGCCCCTAAGCT CCTGATCTATGCTGCGTCCACTTTGCAAACCGGGGTCCCATCAAGG TTC AG CG G CAG TGGATCTGG G AC AG AATTC ACTCTC AC AATCC G C A GCCTG CAGTCTG AAG ATTTTG G AACTTATTACTGTCAG CAG CTTAAA ACTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO: 99 NI-302.63F3-PIMC V _K GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCAATCAGAGTCTTTTTCTA CAGTTCCAACAATAAC AACTACTTAG CTTG GTACCAG CACAAATCCG GACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATC CGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTT CACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATT ACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACC AAGCTGGAGATCAAA SEQ ID NO: 101 NI-302.63F3-PIMC-NSV _K GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCTCACAGAGTCTTTTTCTA CAGTTCCAACAATAAC AACTACTTAG CTTG GTACCAG CACAAATCCG GACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATC CGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTT CACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATT ACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACC AAGCTGGAGATCAAA SEQ ID NO: 103 NI-302.63F3-PIMC-SG V _K GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCAATCAGGGCCTTTTCTA CAGTTCCAACAATAAC AACTACTTAG CTTG GTACCAG CACAAATCCG GACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATC CGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTT CCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATT ACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACC AAGCTGGAGATCAAA SEQ ID NO: 105 NI-302.63F3-PIMC-NQV _K GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCCAACAGAGTCTTTTTCTA CAGTTCCAACAATAAC AACTACTTAG CTTG GTACCAG CACAAATCCG GACAGCCTCCTAAGCTGCTCGTTTACTGGGGATCTACCCGGGAATC CGGGGTCCCTGACCGCTTCAGTGGCAGCGGGTCTGGGACTGACTT CACCATCAGTAGCCTGCAGGCTGAGGATGTTGCAATTTATT ACTGTCACCAATATTATCATAATCCGTACACTTTTGGCCAGGGGACC AAGCTGGAGATCAAA SEQ ID NO: 107 NI-302.35C1-PIMC V _K GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAAAGTGTTGACA ACCAGTTTG CCTG GTACCAACAG AAACCTG G CCAG GCTCCCAG GCT CCTCATTTATGATGCATCCAGGAGGGCCCCTGGCATCCCAGACAGG TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATTAGCA GCCTAGAGC CTGAAGATTTCGCAATTTATTACTGTCAGCATCGTTAC ACCTGG CTCTAC ACTTTTG GCCAG G GG ACC AAG CTG GAG ATC AAA SEQ ID NO: 109

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NI-302.31F11PIMCVk GATATTGTGATGACTCAATCACCACTCTCCCTGCCCGTCGCCCCTG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTATA CAG TAATG G ATACAACTATTTG G ATTG G TACCTG CAG AAG CC AG G G AAGCCTCCACAGCTCCTGGTCTATTTGGGTTCTGATCGGGCCTCCG GGGTCCCCTGACAGGTTCAGTGGCAGTGGATCAGGCAAAGATT TTAC ACTGAACATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTAC TGCATGCAAGGTCTACAAAGTCCGTGGACGTTCGGCCAAGGGACC AAG G TG G AAATC AAA SEQ ID NO: 111 NI-302.2A2-PIMC V _K GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTCTTTTATA CACCTCCAAAAATAAGGACAGTAAGAACTACTTAGGTTGGTACCAG CAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTA CCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTG GGACAGATTTCACTCT CACCATCAGCAGCCTGCAGGCTGAGGATGT GGCAGTTTATTACTGTCAGCAGTATTATACTACTCCTCAGTTCGGCG G AGG G ACC AAG G TG G AAATC AAA SEQ ID NO: 113 NI-302.74C11PIMCVh CAGGTGCAGCTGGTGCAATCTGGGACTGAGGTGCAGAAGCCTGG GGCCTCAGTAAAAGTCTCCTGCAAGGCTTCTGGATACAGTTTCACC GGCTACTTTTTGCACTGGGTACGACAGGCCCCTGGACAAGGGCTT GAGTGGATGGGGTGGATCAACCCTAACAGTGGTGACACAAACTATG CAGAGAAGTTTCGGGGCAGAATCATCATGACCAGGGACACGTCTGT CAGCACAG CCCACATGGAGTTGAGCAGCCTGAGATTTGACGACAC GGCCCTATATTACTGTACGAGAGAGGCCCCTGACCCGGGCGCTGA GACGGACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 115 NI-302.74C11PIMCVl TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGA CAGACGGCCAGGATCACCTGCTCTGGAGATGCAGTGCCAAAGCAG TATATTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTATTCTGG TGATATATAAAGACACTCAGAGGCCTTCAGGGATCCCTGAGCGATT CTCTGGCTCCAACTCAGGGACAACAGTCACGTTGACCATAACTGGC GTCCAGGCAGACGA CGAGGGTGACTATTACTGTCAATCAGCAGACA GTAGTGCTACTTGGGTGTTCGGCGGAGGGACCAAATTGACCGTCCT A SEQ ID NO: 117 NI-302.39G12- PIMCVh GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCACCCTTG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCGTCTCT AATTACGCCATAACTTGGGTCCGCCGGGCTCCAGGGAAGGGGCTG CAATATATTTCAGTAATTTATCGTG ATG G CAG G ACATACTACG G AG A CTCCGTGAGGGGCCGCTTCACCATCTCTAGGGACGATTCCAAGAAC ACTCT CTATCTTCAAATGAACAGCCTGAGATTTGAGGACACGGCTGT GTATTACTGTGCGAGAGCGCACGGCCAATATTACTATGGTGTGGAC GTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 119 NI-302.39G12- PIMCVk GACATCGTGATGACCCAGTCTCCACTCTCCCTGTCCGTCAGCCCT GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTA CATAG TAATG G ATACAACTATTTG G ATTG GTACCG GCAG AAAC CAG GGCAGTCTCCACAGCTCCTGATCTATTTGAGTTCTAATCGGCCCTC CGGGGTCCCTGATAGGTTCAGTGCCAGTGGATCAGGCACAGAGTT CACACTG C AAATCAGCAG AGTG G AG GCTG AG G ATGTTGG G GTTTAT TACTGCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCA AGGTGGAAATCAAA SEQ ID NO: 121 NI-302.11A4-PIMC V _K GAAATTGTGCTGACTCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG GAGAAAGAGCCACCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCA

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GCAGCTACTTCGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAG GCTCCTCATCTATG GTACGTCCCG CAG G GCC ACTG CCATCCCAG AC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCA GCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAACAGTAT GGTAGCTCGTGGACGTTCGGCCCAGGGACCAAGGTGGAAATCAA A SEQ ID NO: 123 NI-302.22H9-PIMC V _K GATATTGTGATGACTCAATCACCACTCTCCCTGTCCGTCAGCCCTG GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACA TAGTAATGGATACAACTATTTGGATTGGTACCGGCAGAAACCAGGG CAGTCTCCACAACTCCTGATCTATTTGAATTCTAATCGGGCCTCCGG GGTCCCTGATAGGTTCAGTGGCAGTGGATCAGGCACAGAGTTCACA CTG TCAG CAG AGTG GAG G CTG AGG ATGTTG GG GTTTATTACT GCATGCAATCTCTGCAAACGTTCACTTTCGGCGGAGGGACCAAGG TGGAAATCAAA SEQ ID NO: 125 NI-302.44D7-PIMC V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC AGCTATGCCATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTCTCAGGTATTGGTTATAGTGATACTAGCACATATTACGC AGACTCCGTGAAGGGCCGCTTCACCGTCTCCAGAGACATTTCCAAG GCTGTATCTGCAATGAATAGCCTGAGGGCCGAGGACACG GCCGTATATTACTGCGCGAAAGGTACCAGGGACTATTACGGTATGG ACGTCTGGGGCCAAGGAACCACGGTCACCGTCTCCTCG SEQ ID NO: 127 NI-302.78H12- PIMCVh CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTC GGGACCCTGTCCCTCACCTGTCTTGTCTCTAGTTACTCCATCAGC AATGGTTACTACTGGGGCTGGATTCGGCAGCCCCCAGGGAAGGGG CTGGAGTGGATTGGGAGTATCTATCATAATGGGAACACCTATTACAA CCCGTCCCTCAAGAGTCGAGTCATCATTTCAGTAGACACGTCCAAG AACCAGTT CTCCCTGAAGTTGAGGTCTGTGACCGCCGCAGACACG GCCGTGTACTACTGTGCGATGCCAAGTGCCACCTATTATTATGGTT CGGGGACTCAATTCCATGCGTTTGATGTCTGGGGCCAAGGGACAA TGGTCACCGTCTCTTCG SEQ ID NO: 129 NI-302.64E5-PIMC V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGG GGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCGAC CAGGCCTGGATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCT GGAGTGGGTTGGCCGGATTAAAACGAAAACTGAGGGTGAAGCAAC AGACTACGCAGCGCCCGTGAGAGGCAGATTCACCATCTCAAGAGAT GATTCAGAAGAC ACGGTGTTTCTGCAAATGAACAGCCTGAAAACCG AGGACACAGCCCTGTATTACTGTACGTCAACGGGAGTCTTAGCAGC AGCTGTCGATGTCTACTGGGGCCAGGGCACCCTGGTCACCGTCTC CTCG SEQ ID NO: 166 NI-302.64E5-PIMC V _K GATATTGTGATGACTCAATCACCAGACTCCCTGGCTGTGTCTCTGG GCGAGAGGGCCACCATGACCTGCAAGTCCAGCCAGAGTCTTTTTCTA CAGTTACAACAATGAGAACTACTTAGCCTGGTATCAGCAGAGACCA GGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAAT CCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATT TCACTCTCAC CATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTA TTACTGTCAGCAATATTATAGTACTCCTCAGACGTTCGGCCAAGGG ACC AAG G TG G AAATCAAA SEQ ID NO: 170

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NI-302.72F10- PIMCVh GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTCGTACAGCCTGG GGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCGGC AGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTG GAGTGGGTGTCAGATATCAGTGGTATTGGTAGTAACACATACTACG CAGACTCCGTGAAGGGCCGTTTCACCATTTCCAGAGACAATTCCGA CAATACGTTG TACCTGGACATGAGCAGCCTGAGAGCCGAGGACAC GGCCAGATATTACTGTGCGAAGGATCGAAAGCGCAGTGGCTGGTA CGAACAGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCG SEQ ID NO: 178 NI-302.72F10- PIMCVk GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGACTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCG CCTACTTAG G CTG GTATCAACAAAAACCTG G CC AG GCTCCC AGGCT CCTCATCTATGATGCATCCATTAGGGCCACTGGCATTCCAGACAGG TTTAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCA GCCTA GAGCCTGAAGATTCTGCAGTTTATTACTGTCACCAGCGTAG CAAGTGGCCTCTTACTTTCGGCGGAGGGACCAAGGTGGAAATCAA A SEQ ID NO: 182 NI-302.12H2-PIMC V _H GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGG GGGGTCCCTGAGACTTTCCTGTGAAGCCTCTGGATTCACCTTTAGC AACTATG CCATGG G CTG GG TCCG CCAG G CTCC AG GG AAGG G G CTG GAGTGGGTCTCAGTAATTAGTGGTACTGGTGGTAGCACATACTACG CAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAT GAACACGCTGTATCTGCAAATGAACAGCCCGAGAGCCGACGACAC GGCCGTATATTACTGTGCGAAAGATCTGAGGAAGATTAGCGGTCCT TTATACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCG SEQ ID NO: 190 NI-302.8M1-PIMC V _H CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGG GGCCTCAGTGAAAGTTTCCTGCAAGGCATCCGGATACACCTTCACC ATCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGAGGAATCAGCCCGAGTGGTGCCCACACAATGTACG CACAGAATTTCCAGGGCAGAGTCACCGTGACCAGGGACACGTCCA CGAGCACAGTC TACATGGAGCTGAGCAGCCTGAGATCTGAGGACA CGGCCGTGTATTACTGTGCGAGAGGGAGCACGGTGACTAACTATC GACCCTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCT CG SEQ ID NO: 196

The present invention also includes fragments of the polynucleotides of the present invention described in various sections herein. In addition, also included in the present invention are polynucleotides that encode hybrid polynucleotides, Fab fragments, and other derivatives as described herein. These polynucleotides can be prepared or produced by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding said antibody can be assembled from chemically synthesized oligonucleotides, for example, as described in Kutmeier et al., BioTechniques 17 (1994), 242; the method, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the antibody coding sequence, annealing and ligation of said oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody or an antigen-binding fragment, variant or derivative thereof can be obtained from a nucleic acid derived from a suitable source. In the event that a clone containing the nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (for example, cDNA libraries of antibodies or libraries derived from antibodies cDNA, or nucleic acid, preferably poly-A ⁺ RNA, isolated from any tissue or cells expressing an HTT-specific antibody, e.g., hybridoma cells selected to express the antibody) by PCR amplification using synthetic primers hybridized to 3' - and the 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library that encodes the antibody. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of an antibody or antigen binding fragment, variant or derivative thereof has been determined, the nucleotide sequence can be processed using nucleotide sequence manipulation techniques well known in the art, for example, using recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current for a description of the techniques. Protocols in Molecular Biology,

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John Wiley & Sons, NY (1998), incorporated by reference herein in its entirety), to produce antibodies having a different amino acid sequence, for example, to introduce amino acid substitutions, deletions and/or insertions.

IV. Antibody polypeptide expression

After processing the isolated genetic material to produce antibodies or antigen binding fragments, variants or derivatives thereof according to the present invention, the polynucleotides encoding said antibodies are typically inserted into an expression vector for introduction into host cells that can be used to produce the required amount of the antibody. Described herein is the recombinant expression of an antibody or a fragment, derivative or analog thereof, eg, an antibody heavy or light chain that binds to a target molecule. After obtaining a polynucleotide encoding an antibody molecule, or an antibody heavy or light chain, or a portion thereof (preferably containing a heavy or light chain variable domain) according to the present invention, a vector for producing the antibody molecule can be prepared using recombinant DNA technology, using techniques well known in the art. Accordingly, methods for producing a protein by expressing a polynucleotide containing an antibody coding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and suitable transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. The present invention accordingly provides replicable vectors containing a nucleotide sequence encoding an antibody molecule of the present invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, WO 86/05807 and WO 89/01036; and US Pat. No. 5,122,464) and the antibody variable domain may be cloned into such a vector to express the full severe or light chain.

The term vector or expression vector as used herein means the vectors used in accordance with the present invention as a vehicle for introducing a desired gene into a host cell and expressing it. As is known to those skilled in the art, such vectors can readily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors suitable for use in the present invention contain a selectable marker, suitable restriction sites to facilitate cloning of the desired gene, and are capable of entering and/or replicating in eukaryotic or prokaryotic cells. Numerous expression vector systems can be used for the purposes of the present invention. For example, one class of vectors uses DNA elements that are derived from animal viruses, such as bovine papillomavirus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others include the use of polycistronic systems with internal ribosome binding sites. In addition, selection of cells into whose chromosomes the DNA has been integrated can be accomplished by introducing one or more markers that allow selection of transfected host cells. The marker may provide prototrophy in an auxotrophic host, resistance to biocides (eg antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to DNA sequences for expression or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers and termination signals.

In specific preferred embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (preferably human) as described above. In one embodiment, this is accomplished using a proprietary expression vector from Biogen IDEC, Inc. called NEOSPLA, as described in US Pat. No. 6,159,730. The vector contains a cytomegalovirus promoter/enhancer, a mouse beta globin core promoter, an SV40 origin of replication, a sequence bovine growth hormone polyadenylation, exon 1 and exon 2 of neomycin phosphotransferase, dihydrofolate reductase gene and leader sequence. This vector was found to provide very high levels of antibody expression when variable and constant region genes were included, transfected into CHO cells, followed by selection on G418-containing media and amplification using methotrexate. Of course, any expression vector capable of providing expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1 and pZeoSV2 ( supplied by Invitrogen, San Diego, CA) and plasmid pCI (60 046697 supplied by Promega, Madison, WI). In general, screening significant numbers of transformed cells to find cells that express acceptably high levels of immunoglobulin heavy and light chains is routine experimental work that can be carried out, for example, in robotic systems. Vector systems are also described in US Pat. No. 5,736,137 and US Pat. No. 5,658,570, each of which is incorporated by reference herein in its entirety. This system provides high expression levels, for example >30 pg/cell/day. Other examples of vector systems are described, for example, in US Pat. No. 6,413,777.

In other preferred embodiments, antibodies or antigen binding fragments, variants or derivatives thereof of the present invention can be expressed using polycistronic constructs such as those described in US Patent Application Published No. 2003-0157641 A1, incorporated herein in its entirety. In these expression systems, multiple gene products of interest, such as antibody heavy and light chains, can be produced from a single polycistronic construct. These systems provide the advantage of using an internal ribosome entry site (IRES) to produce relatively high levels of antibodies. Compatible IRES sequences are described in US Pat. No. 6,193,980, which is also incorporated herein. Those skilled in the art will appreciate that such expression systems can be used to efficiently produce the full range of antibodies described in the present invention. Accordingly, in one embodiment, the present invention provides a vector comprising a polynucleotide that encodes at least a binding domain or variable region of an immunoglobulin chain of an antibody, optionally in combination with a polynucleotide that encodes a variable region of another immunoglobulin chain of said binding molecule.

In general, after obtaining a vector or DNA sequence encoding a monomeric subunit of an antibody, said expression vector can be introduced into a suitable host cell. Introduction of a plasmid into a host cell can be accomplished using various techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection, including lipotransfection using, for example, Fugene® reagent or Lipofectamine, protoplast fusion, calcium phosphate precipitation, DNA-coated cell fusion, microinjection, and intact virus infection. Typically, introduction of the plasmid into the host is carried out using a standard calcium phosphate coprecipitation method. Host cells carrying the expression constructs are cultured under conditions suitable for the production of light chains and heavy chains and analyzed for the synthesis of heavy and/or light chain proteins. Examples of assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorting (FACS), immunohistochemical studies, and the like.

The expression vector is transferred into a host cell using standard techniques, and then the transfected cells are cultured using standard techniques to produce antibodies for use in the methods described herein. Accordingly, the present invention includes host cells containing a polynucleotide encoding an antibody of the present invention, or a heavy or light chain thereof, or at least a binding domain or variable region of a corresponding immunoglobulin, which is preferably operably linked to a heterologous promoter. In an alternative or additional embodiment, the present invention also includes host cells containing a vector as defined hereinabove, comprising a polynucleotide encoding at least an immunoglobulin chain binding domain or variable region of an antibody, optionally in combination with a polynucleotide that encodes the variable region another immunoglobulin chain of said binding molecule. In preferred embodiments, a single vector may be expressed for the expression of double-chain antibodies in a host cell, or vectors encoding both heavy and light chains may be co-expressed to express a complete immunoglobulin molecule, as detailed below.

A host cell can be cotransfected with two expression vectors of the present invention, the first vector encoding a polypeptide derived from the heavy chain and the second vector encoding a polypeptide derived from the light chain. The two vectors may contain identical selectable markers to provide the same level of expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides may be used. In such situations, the light chain is preferably placed before the heavy chain to avoid producing an excess of toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The heavy and light chain coding sequences may comprise cDNA or genomic DNA.

As used herein, the term host cells refers to cells carrying vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of methods for isolating antibodies from recombinant hosts, the terms cell and cell culture are used interchangeably to denote the source of the antibody unless explicitly stated otherwise. In other words, isolating a polypeptide from cells may mean isolating either from pelleted whole cells or from a cell culture containing both medium and suspended cells.

Various host/expression vector systems can be used to express antibody molecules for use in the methods described herein. Such host/expression vector systems include vehicles by which coding sequences of interest can be produced and then purified, but also include cells that can, after transformation or transfection with suitable nucleotide coding sequences, express an antibody molecule of the present invention in situ. These systems include, but are not limited to, microorganisms such as bacteria (eg, Escherichia coli, Bacillus subtilis) transformed with recombinant bacteriophage-based DNA expression vectors, plasmid or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; systems based on insect cells infected with recombinant viral expression vectors (for example, baculovirus) containing antibody coding sequences; systems based on plant cells infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing antibody coding sequences; or systems based on mammalian cells (for example, COS, CHO, NSO, BLK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or mammalian viruses (for example, late adenovirus promoter; vaccinia virus promoter 7.5K). Bacterial cells such as E. coli are preferably used to express the recombinant antibody molecule, and more preferably eukaryotic cells, in particular to express the entire recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with a vector, such as the core promoter element of human cytomegalovirus intermediate-early genes, provide an effective antibody expression system; see, for example, Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 (1990), 2.

Mammalian-derived host cell lines are often used; Those skilled in the art will be able to determine specific preferred host cell lines that are best suited to express the desired gene product. Examples of host cell lines include, but are not limited to, CHO (Chinese hamster ovary cells), DG44 and DUXB11 (Chinese hamster ovary cell lines, DHFR-), HELA (human cervical carcinoma), CVI (monkey kidney line), COS ( CVI-derived line with T-antigen SV40), VERY, BHK (newborn hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblasts) BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney cell line), SP2/O ( mouse myeloma), P3x63Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine epithelial cells), RAJI (human lymphocytes) and 293 (human kidney). In particular, CHO and 293 cells are preferred. Host cell lines are generally available from commercial sources, the American Tissue Culture Collection, or published sources.

In addition, a host cell strain may be selected that modulates the expression of the inserted sequences, or that modulates and processes the gene product in a particular manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Other host cells have properties and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used to provide cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product.

For long-term synthesis of recombinant proteins with high yield, stable expression is preferred. For example, cell lines can be constructed that stably express the antibody molecule. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by suitable expression control elements (eg, promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introducing foreign DNA, the engineered cells can be cultured in an enriched medium for 1-2 days and then transferred to a selective medium. Selectable marker in recombinant

- 62 046697 plasmid imparts resistance to selection to cells, allows stable integration of the said plasmid into chromosomes and ensures growth with the formation of foci, which, in turn, can be cloned and propagated to obtain cell lines. This method may have the advantage of being used to construct cell lines that stably express the antibody molecule.

A number of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase genes (Wigler et al., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. . Sci. USA 48 (1992), 202) and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) in tk-, hgprt- or aprT- cells, respectively. Selection can also be based on resistance to antimetabolites using the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo, which confers resistance to aminoglycoside G-418 (Goldspiel et al., Clinical Pharmacy 12 (1993), 488505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; ); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147). Suitable recombinant DNA technologies generally known in the art are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in chapters 12 and 13 of the source: Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entirety.

Expression levels of the antibody molecule can be increased by vector amplification; For general information, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987). If the marker used in the vector system for antibody expression is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, the level of antibody synthesis increases; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

In vitro synthesis allows scale-up to produce significant quantities of the required polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, for example, in an airlift or continuous stirred tank reactor, or immobilized or entrapped cell cultures, for example, in hollow fibers, microcapsules, agarose microbeads, or in ceramic cartridges. If necessary and/or desired, the polypeptide solutions can be purified using customized chromatographic techniques, for example, gel filtration, ion exchange chromatography, DEAE cellulose chromatography or (immuno)affinity chromatography, for example, after selective biosynthesis of a synthetic hinge region polypeptide, or before/after the hydrophobic interaction chromatography step as described herein.

The genes encoding antibodies or antigen binding fragments, variants or derivatives thereof of the present invention may also be expressed in non-mammalian cells, such as bacterial or insect cells, or yeast or plant cells. Bacteria that readily accept nucleic acids include members of the Enterobacteriaceae, such as E. coli or Salmonella strains; Bacillaceae such as B. subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It should also be borne in mind that when expressed in bacteria, heterologous polypeptides are usually included in inclusion bodies. Heterologous polypeptides must be isolated, purified and then assembled into functional molecules. If tetravalent forms of antibodies are required, the subunits are then assembled into tetravalent antibodies; see, for example, international application WO 02/096948.

When using bacterial systems, a number of preferred expression vectors can be selected based on the intended use of the antibody molecule being expressed. For example, if the synthesis of significant quantities of such a protein is necessary to obtain pharmaceutical compositions with the specified antibody molecule, it may be advisable to use vectors that provide high levels of expression of hybrid protein products that can be easily purified. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody coding sequence can be individually ligated in frame to the lacZ coding region to produce hybrid protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke and Schuster, J. Biol. Chem. 24 (1989), 5503-5509) and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins containing

- 63 046697 glutathione^-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione agarose bead support and subsequent elution in the presence of free glutathione. pGEX vectors are designed to contain protease cleavage sites for thrombin or factor Xa, thereby allowing the cloned target gene product to be separated from the GST fragment.

In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although several other strains, such as Pichia pastoris, are also publicly available. For expression in Saccharomyces, plasmid YRp7 is commonly used, for example (Stinchcomb et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980), 157 ). This plasmid already contains the TRP1 gene, which is a selection marker for a mutant yeast strain that is unable to grow in the absence of tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12). The presence of damaged trpl in the host yeast cell genome in this case provides effective conditions for detection of transformation by cultivation in the absence of tryptophan.

In the Autographa californica insect cell-based system, nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. This virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be individually cloned into minor regions (eg, the polyhedrin gene of the virus) and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

Once an antibody molecule of the present invention is recombinantly expressed, the complete antibodies, dimers thereof, individual light and heavy chains, or other forms of immunoglobulins of the present invention can be purified according to procedures standard in the art, including, for example, using chromatography (e.g., ion exchange, affinity, particularly based on affinity for a specific antigen after protein A chromatography, and size exclusion column chromatography), centrifugation, differential solubility, e.g., ammonium sulfate precipitation, or any other standard purification technique proteins; see, for example, Scopes, Protein Purification, Springer Verlag, N.Y. (1982). A variant of the preferred method for increasing the affinity of antibodies according to the present invention is disclosed in US Patent Publication 2002-0123057 A1. According to one embodiment, accordingly, the present invention also provides a method for producing an anti-HTT antibody or an antibody recognizing mutated and/or aggregated HTT species, and/or fragments thereof, or corresponding immunoglobulin chain(s), wherein said method includes:

(a) culturing a host cell as defined above herein containing a polynucleotide or vector as defined above herein; and (b) isolating said antibody or its corresponding immunoglobulin chain(s) from the culture.

In addition, in one embodiment, the present invention also provides an antibody or corresponding immunoglobulin chain(s) encoded by a polynucleotide as defined hereinabove or produced using said method for producing an anti-HTT antibody, or an antibody that recognizes mutated and/or aggregated HTT species and/or fragments thereof, or the corresponding immunoglobulin chain(s).

V. Hybrid proteins and conjugates

In certain embodiments, an antibody polypeptide comprises a sequence of amino acids, or one or more fragments, not typically associated with an antibody. Examples of modifications are described in more detail below. For example, a single chain Fv fragment of an antibody of the present invention may contain a flexible linker sequence, or may be modified by the addition of a functional moiety (e.g., PEG, drug, toxin, or label such as fluorescent, radioactive, enzyme, nuclear magnetic label, heavy metal and so on.)

The antibody polypeptide of the present invention may comprise, consist essentially of, or be composed of a fusion protein. Hybrid proteins are hybrid molecules that contain, for example, an immunoglobulin HTT-binding domain containing at least one target binding site and at least one heterologous part, i.e. a part with which it is not naturally associated in nature. These amino acid sequences typically may exist in the form of separate proteins that are combined into a hybrid polypeptide; they may also be normally present in one protein but located differently in a hybrid polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by producing and translating a polynucleotide encoding the peptide regions in the desired relative position.

The term heterologous with respect to a polynucleotide or polypeptide means that the specified

- 64 046697 the polynucleotide or polypeptide is derived from an entity other than the rest of the entity being matched. For example, herein, a heterologous polypeptide for connection with an antibody or an antigen binding fragment, variant or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.

As detailed in various sections herein, the antibodies or antigen binding fragments, variants or derivatives thereof of the present invention may also be recombinantly linked to a heterologous polypeptide at the N- or C-terminus, or chemically conjugated (including covalently and non-covalently) to polypeptides or other compounds. For example, antibodies can be recombinantly coupled or conjugated to molecules suitable for use as labels in detection assays and as effector molecules, for example, heterologous polypeptides, drugs, radionuclides or toxins; see, for example, international applications WO 92/08495; WO 91/14438; WO 89/12624; US Patent No. 5314995 and European Patent Application EP 0396387.

Antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention may consist of amino acids linked together by peptide bonds or modified peptide bonds, i.e. peptide isosteres, and may contain amino acids other than the 20 genetically encoded amino acids. Antibodies can be modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are described in detail in primary texts and more comprehensive monographs, as well as in the extensive scientific literature. Modifications can occur in any region of the antibody, including the peptide backbone, amino acid side chains, amino terminus or carboxyl terminus, or moieties such as carbohydrate moieties. It should be understood that the same modification may be present to equal or varying degrees in several regions of a particular antibody. A particular antibody may also contain modifications of many types. Antibodies can be branched, for example as a result of ubiquitination; cyclical; branched or unbranched. Cyclic, branched and branched cyclic antibodies can arise from natural post-translational processes or can be produced using synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent addition of flavin, covalent addition of a heme fragment, covalent addition of a nucleotide or nucleotide derivative, covalent addition of a lipid or lipid derivative, covalent addition of phosphatidylinositol, cross-linking, cyclization, formation of disulfide bonds, demethylation, covalent cross-linking, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristiolation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation-mediated tRNA addition of amino acids to proteins, such as arginylation and ubiquitination; see, for example, Proteins - Structure And Molecular Properties, T. E. Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification Of Proteins, W. C. Johnson, Ed., Academic Press, New York, (1983) 1-12; Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides fusion proteins comprising an antibody, or an antigen binding fragment, variant or derivative thereof, and a heterologous polypeptide. In one embodiment, the fusion protein of the present invention includes, consists essentially of, or consists of a polypeptide having an amino acid sequence corresponding to any one or more VH regions of an antibody of the present invention, or an amino acid sequence corresponding to any one or more VL regions of an antibody of the present invention. the present invention, or fragments or variants thereof, and the sequence of the heterologous polypeptide. In another embodiment, a fusion protein for use in diagnostic and therapeutic methods as described herein comprises, consists essentially of, or consists of a polypeptide having an amino acid sequence corresponding to any one, two, or three of the antibody VH-CDR regions, or fragments thereof, variants or derivatives, or an amino acid sequence corresponding to any one, two, three of the V _L -CDR regions of the antibody, or fragments, variants or derivatives thereof, and the sequence of a heterologous polypeptide. In one embodiment, the fusion protein comprises a polypeptide having the VH-CDR3 amino acid sequence of an antibody of the present invention, or a fragment, derivative or variant thereof, and a heterologous polypeptide sequence, wherein the fusion protein specifically binds to HTT. In another embodiment, the fusion protein comprises a polypeptide having an amino acid sequence corresponding to at least one VH region of an antibody of the present invention and an amino acid sequence corresponding to at least one V _L region of an antibody according to the present invention, or fragments thereof, derivatives or variants, and the sequence of the heterologous polypeptide. Preferably, the VH and VL regions of said fusion protein correspond to a single parent antibody (or scFv or Fab fragment) that specifically binds HTT. In yet another embodiment, a fusion protein for use in diagnostic and therapeutic methods as described herein comprises a polypeptide having an amino acid sequence corresponding to any one, two, three, or more of the VH CDR regions of an antibody, and an amino acid sequence corresponding to any one, two , three or more of the V _L CDR regions of the antibody, or fragments or variants thereof, and the sequence of a heterologous polypeptide. Preferably, two, three, four, five, six or more VH-CDR or VL-CDR regions correspond to one parent antibody (or scFv or Fab fragment) according to the present invention. Nucleic acid molecules encoding these fusion proteins are also covered by the present invention.

Examples of fusion proteins described in the literature include fusion T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531; Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA Cell Biol. USA 9 (1990), 347-353; 667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167) ; CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (1991), 721-730); al., J. Exp. Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991), 1133-1144); . Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886; 1483-1489 (1991); and IgE receptor (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).

As discussed in various sections herein, antibodies or antigen binding fragments, variants or derivatives thereof of the present invention can be coupled to heterologous polypeptides to increase the in vivo half-life of the polypeptides or for use in immunoassays employing methods known in the art. For example, in one embodiment, PEG can be conjugated to antibodies of the present invention to increase their in vivo half-life; see, for example, Leong et al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531 or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

In addition, antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention may be coupled to marker sequences, such as a marker peptide, to facilitate their purification or detection. In preferred embodiments, said amino acid marker sequence is a hexahistidine peptide (HIS), such as the tag in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), as well as other marker sequences, many of which are commercially available available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for example, hexahistidine allows for convenient purification of the fusion protein. Other peptide tags suitable for purification include, but are not limited to, the HA tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767), glutathione S-transferase (GST), c- myc and FLAG marker; see, for example, a review of epitope tagging techniques in Bill Brizzard, BioTechniques 44 (2008) 693-695, the contents of which are expressly incorporated herein by reference; in table 1 on page 694 shows the most common epitope tags suitable for use in the present invention.

Fusion proteins can be prepared using methods well known in the art; see, for example, US Pat. No. 5,116,964 and US Pat. No. 5,225,538. The particular junction site may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression, which is performed as described above.

Antibodies of the present invention may be used in unconjugated form or may be conjugated to at least one of a number of molecules, for example, to improve the therapeutic characteristics of the molecule, to facilitate target detection, or for imaging or therapy in a patient. Antibodies or antigen binding fragments, variants or derivatives thereof of the present invention may be labeled or conjugated either before or after purification, if purification is carried out. In particular, the antibodies or antigen binding fragments, variants or derivatives thereof of the present invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents or PEGs.

Immunotoxin conjugates comprising standard antibodies are widely described in the art. These toxins can be conjugated to antibodies using standard conjugation techniques; or immunotoxins containing toxic protein moieties can be synthesized as fusion proteins. The antibodies of the present invention can be suitably used to produce such immunotoxins.

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Illustrative examples of such immunotoxins are described in the sources: Byers, Seminars Cell. Biol.

(1991), 59-70 and Fanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates can also be prepared using various techniques depending on the conjugation agent chosen. For example, biotin conjugates are prepared by reacting, for example, an HTT binding polypeptide with an activated biotin ester, such as biotin-H-hydroxysuccinimide ester. Likewise, fluorescent marker conjugates can be prepared in the presence of a coupling agent, such as those listed herein, or by reaction with an isothiocyanate, preferably fluorescein isothiocyanate. Conjugates of these antibodies or antigen-binding fragments, variants or derivatives thereof according to the present invention are prepared in a similar manner.

The present invention also includes antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention conjugated to a diagnostic or therapeutic agent. These antibodies can be used diagnostically, for example, to demonstrate the presence of HTT amyloidosis, to determine the risk of a disease or disorder associated with mutated and/or aggregated HTT, to monitor the development or progression of such a disease, i.e. a disease in which the occurrence of aggregated NTT or associated with aggregated NTT is observed, or as part of a clinical trial procedure, for example, to determine the effectiveness of a particular treatment and/or prophylaxis regimen. According to one embodiment, accordingly, the present invention relates to a labeled antibody with detection capability. Moreover, according to one embodiment, the present invention provides an antibody that is attached to a drug. Detection may be facilitated by coupling of an antibody or an antigen-binding fragment, variant or derivative thereof to a detectable substance. The detectable substances or label may generally be represented by an enzyme; heavy metal, preferably gold; a dye, preferably a fluorescent or luminescent dye; or a radioactive label. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positronic metals for various positron emission tomography applications, and non-radioactive paramagnetic metal ions; see, for example, US Pat. No. 4,741,900 for a description of metal ions that can be conjugated to antibodies for diagnostic use in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of the luminescent material include luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, 'in or ⁹⁹ Tc. Accordingly, according to one embodiment, the present invention provides a detectably labeled antibody, characterized in that the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, and a heavy metal.

The antibody or antigen binding fragment, variant or derivative thereof may also be detectably labeled by coupling with a chemiluminescent compound. The presence of the chemiluminescent-labeled antibody is then determined by detecting the luminescence produced by the chemical reaction. Examples of compounds suitable for use as chemiluminescent labels include, but are not limited to, luminol, isoluminol, aromatic acridine ester, imidazole, acridine salt and oxalic acid ester.

One method for detectably labeling an antibody or an antigen-binding fragment, variant or derivative thereof is to link it to an enzyme and use the linked enzyme-linked immunosorbent assay (ELISA) product (Voller, A., The Enzyme Linked Immunosorbent Assay (ELISA) Microbiological Associates Quarterly Publication, Walkersville , Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme coupled to the antibody reacts with a suitable substrate, preferably a chromogenic substrate, such that a chemical moiety is formed which can be detected, for example, using spectrophotometric, fluorimetric or visual methods. Enzymes that can be used for detectable labeling of antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In addition, detection can be carried out using colorimetric methods involving the use of a chromogenic enzyme substrate. Detection can also be accomplished by visual comparison of the depth of enzymatic reaction of the substrate with comparison with similarly prepared standards.

Detection can also be carried out using any of a variety of other immunoassays. For example, by radiolabeling an antibody or an antigen-binding fragment, variant, or derivative thereof, detection of the antibody using a radioimmunoassay (RIA) is possible (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), incorporated herein by reference). The radioactive isotope may be detected using, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

The antibody or antigen binding fragment, variant or derivative thereof may also be detectably labeled using fluorescent metals such as ¹⁵² Eu or other lanthanides. These metals can be attached to the antibody through metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various fragments to an antibody or an antigen binding fragment, variant or derivative thereof are well known, see, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc. ., (1987) 623-53; Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al (eds.), (1985) 475- 506; Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy, in: Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al (eds.), Academic Press (1985) 303-16; Thorpe et al., The Preparation And Cytotoxic Properties of Antibody-Toxin Conjugates, Immunol Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances the stability or effectiveness of a binding molecule, such as a binding polypeptide, such as an antibody or an immunospecific fragment thereof, can be conjugated. For example, in one embodiment, PEG may be conjugated to the binding molecules of the present invention to increase their in vivo half-life. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and methods of use

The present invention relates to a composition containing the above-mentioned HTT binding molecule, for example, an antibody or an antigen binding fragment thereof according to the present invention, or a derivative or variant thereof, or a polynucleotide, vector or cell provided by the present invention, as defined above herein. In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition of the present invention may contain additional agents, such as interleukins or interferons, depending on the intended use of the pharmaceutical composition. For use in the treatment of a disease or disorder in which the occurrence of mutated and/or aggregated HTT is observed, or associated with mutated and/or aggregated HTT, such as HTT amyloidosis, the additional agent may be selected from the group consisting of small organic molecules, antibodies to NTT; and their combinations. Thus, in a particular preferred embodiment, the present invention relates to the use of an HTT binding molecule, e.g., an antibody or an antigen binding fragment thereof according to the present invention, or a binding molecule having substantially identical binding specificity to any of the above, a polynucleotide, a vector or a cell according to the present invention for the preparation of a pharmaceutical or diagnostic composition for the prophylactic and therapeutic treatment of Huntington's disease (HD), and/or a disease or disorder associated with NTT and/or NTT amyloidosis, monitoring the progression of HD and/or a disease or disorder associated with NTT and/or NTT amyloidosis, or the response to treatment for NTT amyloidosis in a subject, or to determine the subject's risk of developing a disease or disorder associated with NTT.

Thus, in one embodiment, the present invention provides a method of treating a disease or disorder characterized by abnormal accumulation and/or deposition of NTT, and/or aggregated and/or mutated NTT in affected systems and organs, comprising administering to a subject in need thereof a therapeutically effective amount any of the above-described HTT-binding molecules, antibodies, polynucleotides, vectors or cells according to the present invention.

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A particular advantage of the therapeutic approach proposed in the present invention lies in the fact that the recombinant antibodies of the present invention are derived from B cells or memory B cells of healthy human subjects without signs or symptoms of disease, for example, carriers of an asymptomatic mutation and/or mutations with which exhibits the occurrence of aggregated NTT, or is associated with aggregated NTT, and is therefore likely to be capable of preventing the clinical manifestations of a disease associated with the mutated and/or aggregated NTT, or reducing the risk of developing a clinical manifestation of a disease or disorder, or delay the onset or progression of clinical manifestations of a disease or disorder. Typically, the antibodies of the present invention have also successfully undergone somatic maturation, i.e. optimization of selectivity and efficiency due to high binding affinity of the target HTT molecule through somatic mutations of the variable regions of the antibody.

The fact that in vivo, for example in humans, such cells were not activated by congeners or other physiological proteins or cellular structures to cause an autoimmune or allergic reaction is also of great medical significance, since it indicates a significant increase in the likelihood of successful passage through the clinical trial phases. Efficacy, acceptability and tolerability may be said to have already been demonstrated prior to preclinical and clinical development of a prophylactic or therapeutic antibody in at least one human subject. Thus, it can be expected that specific efficacy against target structures when used as a therapeutic agent, along with a decrease in the likelihood of side effects, significantly increases the clinical likelihood of successful use of human-derived anti-HTT antibodies according to the present invention.

The present invention also provides a pharmaceutical and diagnostic kit or kit, respectively, containing one or more containers filled with one or more of the above-described ingredients, for example, an anti-HTT antibody, a binding fragment, derivative or variant thereof, a polynucleotide, a vector or a cell according to the present invention. Such container(s) may be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, evidencing approval by that agency of manufacture, use, or sale for administration to humans. In an alternative or additional embodiment, the kit contains reagents and/or instructions for use in appropriate diagnostic assays. The composition, for example the kit according to the present invention, is of course suitable, in particular, for risk assessment, diagnosis, prevention and treatment of Huntington's disease and/or a disease or disorder which is characterized by the presence of mutated and/or aggregated HTT, and, in particular, applicable to the treatment of disorders typically characterized by NTT amyloidosis.

Formulations of the pharmaceutical compositions of the present invention can be prepared in accordance with methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) (University of Sciences in Philadelphia, ISBN 0-683306472). Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, wetting agents of various types, sterile solutions, and the like. Compositions containing such carriers can be formulated using well known standard methods. Said pharmaceutical compositions may be administered to a subject at a suitable dose. Administration of suitable compositions can be accomplished in a variety of ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, intranasally, topically or intradermally, or by delivery through the spinal cord or brain. Aerosol formulations, such as nasal sprays, include purified aqueous or other solutions of the active agent with preservative and isotonic agents. Such formulations are preferably adjusted to isotonicity and pH values compatible with the mucous membranes of the nasal cavity. Formulations for rectal or vaginal administration may be presented as suppositories in a suitable carrier.

The dosage regimen is determined by the attending physician based on clinical factors. As is well known in the medical field, dosages for any particular patient depend on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and concurrent administration of other medicines. A typical dose may, for example, be from 0.001 to 1000 μg (or correspond to the amount of nucleic acid to express or inhibit expression in the specified range); however, doses beyond these illustrative ranges are included, particularly taking into account the above factors. Typically, doses may vary, for example, from about 0.0001 to 100 mg/kg, more commonly from 0.01 to 5 mg/kg (eg, 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.) body weight of the host. For example, the dosage may be 1 mg/kg body weight or 10 mg/kg body weight, or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Intermediate doses within these ranges are also within the scope of the present invention. Such doses may be administered to subjects daily, over

- 69 046697 day, weekly or in accordance with any other scheme determined by empirical analysis. Examples of treatment include administering multiple doses over an extended period of time, for example, over at least six months. Additional examples of treatment regimens include once every two weeks or once a month, or once every 3 to 6 months. Examples of dosing regimens include 1-10 or 15 mg/kg on consecutive days, 30 mg/kg every other day, or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within specified ranges. Progress can be monitored through periodic evaluation. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Bases for parenteral use include sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. IV bases include fluid and nutrient replenishment agents, electrolyte replenishers (eg, dextrose Ringer's solution), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases, and the like. In addition, the pharmaceutical composition of the present invention may contain additional agents, such as dopamine or psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

In addition, according to a preferred embodiment of the present invention, the specified pharmaceutical composition can be included in the vaccine, for example, in the case where the pharmaceutical composition according to the present invention contains an antibody to HTT or an HTT binding fragment, derivative or synthetic or biotechnological variant, for passive immunization . As mentioned in the Background Art section, mutated and/or aggregated HTT species, and/or fragments or derivatives thereof, are the main trigger of HTT amyloidosis. Accordingly, it is reasonable to expect that passive immunization with the human anti-HTT antibodies and equivalent HTT-binding molecules of the present invention will circumvent some of the adverse events associated with active immunization therapy options and provide a reduction in HTT aggregation. Accordingly, the proposed anti-HTT antibodies and their equivalents according to the present invention are particularly suitable for use as a vaccine to prevent or alleviate diseases or disorders in which the presence of aggregated HTT is observed, or caused by aggregated HTT, for example, HD.

In one embodiment, it may be advantageous to use recombinant Fab fragments (rFab) and single chain fragments (scFv) of the antibody of the present invention, which can more easily penetrate the cell membrane. For example, Robert et al., Protein Eng. Des. Sel. (2008); S1741-0134, previously published on the Internet, describes the use of hybrid recombinant Fab fragments (rFab) and single chain fragments (scFv) of the monoclonal antibody WO-2, which recognizes an epitope in the N-terminal region of Abeta. The engineered fragments were able to: (i) prevent the formation of amyloid fibrils, (ii) disaggregate already formed Abeta1-42 fibrils, and (iii) inhibit Abeta1-42 oligomer-mediated neurotoxicity in vitro, as effectively as the full-length IgG molecule. The obvious advantages of using engineered small Fab and scFv antibodies lacking effector function include more efficient penetration of the blood-brain barrier and minimizing the risk of promoting inflammatory adverse reactions. In addition, while maintaining the binding specificity of full-length antibodies, scFv and single-domain antibodies can be expressed as individual genes and intracellularly in mammalian cells as intrabodies, opening the possibility of altering the folding, interactions, modification, or subcellular localization of their targets; For review information, see, for example, Miller and Messer, Molecular Therapy 12 (2005), 394-401.

According to another approach described in the source: Muller et al., Expert Opin. Biol. Ther. (2005), 237-241, uses a technology platform called superantibody technology, which is reported to allow the introduction of antibodies into living cells without damaging them. Such cell-penetrating antibodies open up new diagnostic and therapeutic possibilities. For these antibodies, the term monoclonal transbodies was adopted.

In a further embodiment, it may be advisable to co-administer or sequentially administer other antibodies suitable for treating a disease, disorder or symptom associated with the occurrence of mutated and/or aggregated HTT. In one embodiment, said additional antibody is included in the pharmaceutical composition of the present invention. Examples of antibodies that can be used for treatment

- 70 046697 subject, include, but are not limited to, antibodies targeting CD33, SGLT2, IL-6 and

IL-1.

In a further embodiment, it may be advisable to co-administer or sequentially administer other agents suitable for treating the disease, disorder or symptoms associated with the mutated and/or aggregated HTT. According to one embodiment, said additional agent is included in the pharmaceutical composition of the present invention. Examples of agents that may be used to treat a subject include, but are not limited to: VMAT2 inhibitors targeting involuntary muscle movements such as Xenazine™, anti-inflammatory agents such as diflunisal, corticosteroids, 2-(2,6-dichloroanilino )phenylacetic acid (diclofenac), isobutylphenylpropanoic acid (ibuprofen); diuretics, epigallocatechin gallate, melphalan hydrochloride, dexamethasone, bortezomib, bortezomib-melphalan, bortezomib-dexamethasone, melphalan-dexamethasone, bortezomibmelphalan-dexamethasone; antidepressants, antipsychotic drugs, antipsychotics, anti-dementia drugs (for example, the NMDA receptor antagonist memantine), acetylcholinesterase inhibitors (for example, donepezil hydrochloride, rivastigmine, galantamine), glutamate receptor antagonists and other nootropics, drugs for correcting blood pressure (for example, dihydralazine , methyldopa), cytostatics, glucocorticoids, angiotensin-converting enzyme (ACE) inhibitors; anti-inflammatory agents or any combination thereof.

A therapeutically effective dose or amount refers to an amount of the active ingredient sufficient to relieve the symptoms or condition. The therapeutic efficacy and toxicity of such compounds can be determined by performing standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining the ED50 (dose therapeutically effective in 50% of the population) and LD50 (dose lethal in 50% of the population). The ratio of doses producing therapeutic and toxic effects is the therapeutic index, and can be expressed as the LD50/ED50 ratio.

From the foregoing, it is apparent that the present invention covers any use of an HTT binding molecule and/or fragments thereof comprising at least one CDR region of the above-described antibody, particularly for the diagnosis and/or treatment of a disease or disorder associated with mutated and/or aggregated species NTT, and/or fragments thereof as described above, for example, HD and/or NTT amyloidosis. Preferably, said binding molecule is an antibody according to the present invention or an immunoglobulin chain corresponding thereto. In addition, the present invention relates to anti-idiotypic antibodies to any of the antibodies mentioned above herein. These are antibodies or other binding molecules that bind to a unique antigenic peptide sequence located in the antibody variable region near the antigen binding site and are suitable, for example, for detecting anti-HTT antibodies in a sample obtained from a subject. According to one embodiment, accordingly, the present invention provides an antibody as defined above and as defined below, or an HTT binding molecule having substantially the same binding specificity as any of the above, a polynucleotide, a vector, or a cell as defined herein. as defined herein, or a pharmaceutical or diagnostic composition containing any of the foregoing, for use in prophylactic treatment, therapeutic treatment and/or in monitoring the progression or response to treatment of a disease or disorder associated with NTT, wherein said disorder is preferably associated with NTT amyloidosis, for example, Huntington's disease (HD).

According to another embodiment, the present invention relates to a diagnostic composition containing any of the above-described HTT-binding molecules, antibodies, antigen-binding fragments, polynucleotides, vectors or cells according to the present invention and, optionally, suitable detection means, such as reagents commonly used in immunodiagnostics or nucleic acid-based diagnostic methods. The antibodies of the present invention are suitable, for example, for use in immunoassays, where they can be used in liquid phase or bound to a solid phase carrier. Examples of immunoassays in which the antibody of the present invention can be used include competitive and non-competitive immunoassays in direct or indirect format. Examples of such immunoassays include radioimmunoassay (RIA), sandwich assay (immunometric assay), flow cytometry and Western blotting. The antigens and antibodies of the present invention can be associated with a variety of different carriers and can be used to isolate cells specifically associated with them. Examples of well-known supports include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses and magnetite. For purposes of the present invention, the carrier may be soluble or insoluble in nature. There are many different labels and labeling methods known to those skilled in the art. Examples of the types of labels suitable for use in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds; see also the implementations discussed above.

In a further embodiment, the HTT-binding molecules, particularly the antibodies, of the present invention may also be used in a method of diagnosing a disease or disorder in an individual by obtaining a sample of a body fluid of the individual being tested, which may be a blood sample, a plasma sample, a serum sample, a lymph or any other body fluid sample, such as a saliva or urine sample, and contacting said body fluid sample with an antibody of the present invention under conditions such that the antibody is complexed with the antigen. The level of such complexes is then determined using methods known in the art; in this case, a level significantly higher than the level of formation in the control sample indicates the presence of a disease or disorder in the tested individual. The specific antigen bound by the antibodies of the present invention can be used in the same manner. Accordingly, the present invention relates to an in vitro immunoassay involving a binding molecule, for example, an antibody or antigen binding fragment thereof according to the present invention.

In a further embodiment of the present invention, the HTT-binding molecules, in particular the antibodies, of the present invention may also be used in a method of diagnosing a disease or disorder in an individual by obtaining a biopsy specimen from the individual being tested.

In this context, the present invention also relates to a method specifically designed for this purpose. For example, an antibody chip may be used that is, for example, loaded with antibodies or equivalent antigen-binding molecules of the present invention that specifically recognize HTT. An overview of the design of immunoassays with microarrays is given in the source: Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696. Accordingly, the present invention also relates to microchips loaded with HTT binding molecules in accordance with the present invention.

In one embodiment, the present invention provides a method for diagnosing a disease or disorder associated with mutated and/or aggregated HTT species, and/or fragments thereof, in a subject, the method comprising detecting the presence of HTT, and/or mutated and/or aggregated HTT in a sample from a subject being diagnosed using at least one antibody of the present invention, an HTT binding fragment thereof, or an HTT binding molecule having a binding specificity substantially identical to any of the above, wherein the presence of a pathologically mutated and/or aggregated NTT indicates HD and/or NTT amyloidosis, and an increase in the level of pathologically mutated and/or aggregated NTT relative to the level of physiological NTT indicates progression of GD and/or NTT amyloidosis in the specified subject.

The subject being diagnosed may have asymptomatic disease or preclinical disease. Preferably, the control subject has a disease associated with mutated and/or aggregated HTT, such as Huntington's disease (HD), wherein a level of pathologically mutated and/or aggregated HTT similar to the reference standard indicates that the subject will be diagnosed NTT amyloidosis or there is a risk of developing NTT amyloidosis. In an alternative or additional embodiment, a control subject who does not have NTT amyloidosis is selected as the second control subject, wherein a level of physiological NTT and/or mutated and/or aggregated NTT different from the reference standard indicates that the subject will be diagnosed with NTT -amyloidosis or there is a risk of developing NTT amyloidosis. Preferably, the subject being diagnosed and the control subject(s) are of the same age group. The sample for analysis can be any body fluid that is expected to contain pathologically mutated and/or aggregated HTT, for example, blood, blood plasma, serum, urine, peritoneal fluid, saliva or cerebrospinal fluid (CSF). According to another aspect of the present invention, the antibodies of the invention can be used in the detection of soluble and aggregated HTT by, for example, a time-resolved fluorescence resonance energy transfer (TR-FRET) duplex immunoassay as described in Baldo et al., Chem. Biol. 19(2) (2012), 264-275, the contents of which, in particular the experimental procedures on pp. 273-274, are incorporated herein.

In addition, as described, for example, in Ren et al., Nature Cell Biol. 11 (2) (2009), 219225, mammalian cells are able to internalize fibrillar polyglutamine peptide aggregates in culture, which enter the cytosolic compartment and are sequestered into aggresomes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. These internalized fibrillar aggregates were able to selectively recruit soluble cytoplasmic proteins and determine the heritable phenotype of cells expressing a homologous amyloidogenic protein from a chromosomal locus. Accordingly, according to one embodiment of the present invention, an anti-HTT antibody is capable of reducing the extracellular spread or transneuronal spread of toxic NTT species, as demonstrated by Pecho-Vriesling et al. Nat. Neurosci. (2014) doi:10.1038/nn.3761 for huntingtin or other proteins involved in neurodegeneration such as α-synuclein; see for example Guo et al., Nat Med. 20(2) (2014), 130-138.

The level of physiological HTT, and/or pathologically mutated and/or aggregated HTT can be assessed using any suitable method known in the art, including, for example, HTT analysis using one or more techniques selected from Western blotting, immunoprecipitation, solid phase enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectrometry (MS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF), enhanced surface laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF), high-performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS) and laser densitometry. Preferably, said in vivo imaging of the ITT includes scintigraphy, positron emission tomography (PET), single photon emission tomography (SPET), near-infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).

One embodiment respectively provides an antibody of the present invention or an HTT binding molecule having binding specificity substantially identical to any of the above, a polynucleotide, vector or cell as defined herein above, or a pharmaceutical or diagnostic composition comprising either of the above for use in the prophylactic treatment, therapeutic treatment and/or monitoring of progression or response to treatment of a disease or disorder associated with NTT. Accordingly, the present invention also generally relates to a method for diagnosing or monitoring the progression of a disease or disorder associated with NTT (such as NTT amyloidosis) in a subject, the method comprising determining the presence of NTT in a sample from the subject being diagnosed using at least at least one antibody of the present invention or an HTT-binding molecule having binding specificity substantially identical to any of the above, wherein the presence of mutated, misfolded and/or aggregated HTT species or fragments thereof is indicative of a disease or disorder. In one embodiment, a method is provided for diagnosing or monitoring the progression of HTT amyloidosis in a subject, comprising determining the presence of mutated and/or aggregated HTT, and/or fragments thereof, in a sample from a subject being diagnosed using at least one antibody of the present invention. or an HTT-binding molecule characterized by substantially identical binding specificity to any of the above, wherein the presence of mutated and/or aggregated HTT and/or a fragment thereof indicates presymptomatic, prodromal or clinical HTT amyloidosis, an increase in the level of HTT aggregates compared to level of physiological NTT or compared with a reference sample derived from a healthy control subject, or a control sample from the same subject, indicates progression of presymptomatic, prodromal or full-blown NTT amyloidosis. One skilled in the art will appreciate that, in one embodiment, the method is also used to diagnose or monitor the progression of any other disease or disorder from the group of disorders associated with NTT, as defined above herein.

As noted above, the antibodies of the present invention, fragments and molecules thereof, having the same binding specificity as said antibodies and fragments thereof, can be used not only in vitro, but also in vivo, and in addition to diagnostic use, therapeutic options can also be implemented. applications. According to one embodiment, accordingly, the present invention also provides an HTT-binding molecule comprising at least one CDR region of an antibody according to the present invention, for the preparation of a composition for in vivo detection or targeting of a therapeutic and/or diagnostic agent to HTT in a human body or animal. Potential therapeutic and/or diagnostic agents may be selected from the non-exhaustive lists of therapeutic agents suitable for the treatment of NTT amyloidosis and potential labels as described above. With respect to in vivo imaging, in one preferred embodiment, the present invention provides said HTT binding molecule comprising at least one CDR region of an antibody of the present invention, wherein in vivo imaging includes scintigraphy, positron emission tomography (PET), single photon emission tomography ( OFET), optical vi

- 73 046697 visualization in the near infrared region of the spectrum (NIR) or magnetic resonance imaging (MRI). According to a further embodiment, the present invention also provides said HTT binding molecule comprising at least one CDR region of an antibody of the present invention, or said molecule for the preparation of a composition for the above-described in vivo imaging methods, for use in a method for diagnosing or monitoring a subject progression of a disease or disorder associated with NTT, as defined above herein.

VII. Peptides with epitopes specific for HTT aggregates

In a further aspect, the present invention provides peptides containing an epitope of the polyproline-rich region of HTT specifically recognized by any antibody of the present invention. Preferably, such a peptide contains or consists of the amino acid sequence set forth in SEQ ID No.: 146, 147, 148, 149, 150, 152, 153, 155, 156, 139, 151, 154, 158, 161, 157, 159, 160, as a unique linear epitope recognized by the antibody, or a modified sequence thereof, in which one or more amino acids are replaced, deleted and/or added, and the specified peptide is recognized by any antibody according to the present invention, preferably the antibody NI-302.74C11, NI- 302.15F9, NI-302.39G12, NI-3O2.11A4, NI302.22H9, NI-302.37C12, NI-302.55D8, NI-302.78H12, NI-302.71F6, NI-302.33G11, NI-302.44D7, 302. 7A8, NI-302.3D8, NI-302.46C9, N1-302.11H6, NI-302.18A1, NI-302.52C9 and/or NI-302.8F1.

In a further aspect, the present invention provides peptides containing an epitope of the proline-rich region of HTT specifically recognized by any antibody of the present invention. Preferably, such a peptide contains or consists of the amino acid sequence shown in SEQ ID No: 140, 141, 142, 143, 200, as a unique linear epitope recognized by the antibody, or a modified sequence thereof in which one or more amino acids are replaced, removed and/or added, and the specified peptide is recognized by any antibody according to the present invention, preferably the antibody NI-302.63F3, NI-302.31F11, NI-302.2A2, NI302.15D3 and/or NI-302.64E5.

Additionally, in one embodiment, the present invention provides peptides containing an epitope of the C-terminal region of HTT that is specifically recognized by any antibody of the present invention. Preferably, such a peptide contains or consists of the amino acid sequence set forth in SEQ ID NO: 145 or SEQ ID NO: 202 as a unique linear epitope recognized by the antibody, or a modified sequence thereof in which one or more amino acids are replaced, deleted, or /or added, and the specified peptide is recognized by any antibody according to the present invention, preferably the antibody NI302.35C1 or NI-302.72F10.

In a further aspect, the present invention provides peptides containing an epitope of the N-terminal region of HTT specifically recognized by any antibody of the present invention. Preferably, such a peptide contains or consists of the amino acid sequence set forth in SEQ ID NO: 144 as a unique linear epitope recognized by the antibody, or a modified sequence thereof in which one or more amino acids are replaced, deleted and/or added, wherein said peptide recognized by any antibody of the present invention, preferably the NI-302.15E8 antibody.

Additionally, in one embodiment, the present invention provides peptides containing an HTT Q/P-rich region epitope specifically recognized by any antibody of the present invention. Preferably, such a peptide contains or consists of the amino acid sequence set forth in SEQ ID NO: 201 as a unique linear epitope recognized by the antibody, or a modified sequence thereof in which one or more amino acids are replaced, deleted and/or added, wherein said peptide recognized by any antibody of the present invention, preferably the NI-302.7D8 antibody.

In addition, according to one embodiment, the present invention provides peptides containing an HTT epitope specifically recognized by any antibody of the present invention, preferably the antibody NI-302.6N9, NI-302.12H2, NI-302.8M1 and/or NI-302.4A6, in in which one or more amino acids are replaced, deleted and/or added, and the specified peptide is recognized by any antibody according to the present invention.

In one embodiment of the present invention, such a peptide may be used to diagnose or monitor a disease or disorder associated with mutated HTT species, misfolded HTT species, and/or aggregated HTT species, and/or fragments thereof, in a subject, such as HD and/or HTT -amyloidosis, including the step of determining the presence of an antibody that binds to a peptide in a biological sample from the specified subject, and for use in the diagnosis of such disease in the specified subject by measuring the levels of antibodies that recognize the above-described peptide according to the present invention and comparing the results of said measurements with levels found in healthy subjects of the appropriate age and sex. Accordingly, according to one embodiment, the present invention relates to a method for diagnosing

- 74 046697 for the diagnosis of NTT amyloidosis indicating presymptomatic or clinical HD in a subject, comprising the step of determining the presence of an antibody that binds to a peptide as defined above in a biological sample from said subject. In this method, an elevated level of a measured antibody specific for a peptide of the present invention indicates that the subject has a diagnosis of presymptomatic or clinical GD or that the subject has any other disease or disorder from the group of disorders associated with ITT as defined above in this document. The peptide of the present invention can be included in a chip, kit and composition, respectively, as described above herein. In this context, the present invention also provides a kit suitable for diagnosing or monitoring the progression of HD and/or HTT amyloidosis, comprising at least one antibody of the present invention or an HTT binding molecule having substantially the same binding specificity as that - any of the above, a polynucleotide, vector or cell and/or peptide as defined above herein, optionally with reagents and/or instructions for use.

The above description describes the present invention in general terms. Unless otherwise noted, terms in this document have the definitions provided in the Oxford Dictionary of Biochemistry and Molecular Biology (Oxford University Press, 1997, revised 2000, reprint 2003, ISBN 0198506732). A number of documents are cited throughout this specification. Full bibliographic data is given at the end of this description, immediately before the claims. The contents of all references cited (including publications, issued patents, published patent applications cited in the text of this application, including in the technical description section, manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference; however, this does not mean that any document cited is actually prior art with respect to the present invention.

A better understanding may be gained by reference to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Example 1. Isolation and identification of antibodies to HTT

Human-derived antibodies targeting HTT and/or mutated and/or aggregated HTT species and/or fragments thereof were identified using the method described in international application WO 2008/081008, the contents of which are incorporated herein by reference , with modifications. In particular, wild-type HTT proteins and mutant HTT proteins produced through recombinant expression have been used in both native and mutated/aggregated conformational forms to identify HTT-targeting antibodies. Mutated/aggregated conformational forms were obtained in vitro using a procedure similar to that described in Example 3.

Example 2 Antibody Sequencing

The amino acid sequences of the variable regions of the anti-HTT antibodies described above were determined based on their mRNA sequences, see FIG. 1. Briefly, live B cells were collected from selected cultures of non-immortalized memory B cells. The mRNA was then extracted from cells producing the selected anti-HTT antibodies, converted to cDNA, and the sequences encoding the antibody variable regions were amplified by PCR, cloned into plasmid vectors, and sequenced.

Briefly, a combination of primers corresponding to the sequences of all immunoglobulin families of the human germline repertoire was used to amplify the leader peptides, V segments and J segments. The first round of amplification was performed using leader peptide-specific primers at the 5' end and constant region-specific primers at the 3' end (Smith et al., Nat Protoc. 4 (2009), 372-384). For heavy chains and kappa light chains, a second round of amplification was performed using V-specific primers at the 5' end and J-specific primers at the 3' end. For lambda light chains, a second round of amplification was performed using V-segment-specific primers at the 5' end and C-region-specific primers at the 3' end (Marks et al., Mol. Biol. 222 (1991) , 581-597; de Haard et al., J. Biol. 26 (1999), 18218-18230).

Identification of an antibody clone with the required specificity was accomplished by repeat ELISA screening after recombinant expression of the complete antibodies. Recombinant expression of full human IgG1 antibodies was achieved by inserting variable heavy and light chain sequences into the correct reading frame of expression vectors, complementing the variable region sequence with a sequence encoding the leader peptide at the 5' end and a sequence encoding the appropriate constant domain(s). s) at the 3' end. For this purpose, primers were designed containing restriction sites to facilitate cloning

- 75 046697 synthesis of variable sequences of heavy and light chains into expression vectors with antibodies. Immunoglobulin heavy chains were expressed by inserting the immunoglobulin heavy chain RT-PCR product in-frame into a heavy chain expression vector carrying the signal peptide and human or mouse immunoglobulin gamma 1 constant domains. Immunoglobulin kappa light chains were expressed by inserting the kappa light chain RT-PCR product in-frame into a light chain expression vector providing the signal peptide and human immunoglobulin kappa light chain constant domain. Immunoglobulin lambda light chains were expressed by inserting the lambda light chain RT-PCR product in-frame into a lambda light chain expression vector providing the signal peptide and human or mouse immunoglobulin lambda light chain constant domain.

Functional recombinant monoclonal antibodies were produced by co-transfecting HEK 293 or CHO cells (or any other suitable recipient cell line of human or mouse origin) with an Ig heavy chain expression vector and an Ig kappa or lambda light chain expression vector. The recombinant human monoclonal antibody was then isolated from the conditioned medium by standard protein A column purification. The recombinant human monoclonal antibody can be produced in unlimited quantities using transiently or stably transfected cells. Stable cell lines producing recombinant human monoclonal antibody can be generated either directly using Ig-carrying expression vectors or by re-cloning Ig variable regions into different expression vectors. Derivatives such as F(ab), F(ab)2 and scFv can also be obtained from these Ig variable regions. Framework and complementarity determining regions were determined by comparison with reference antibody sequences available in databases such as Abysis (https://www.bioinf.org.uk/abysis/) and https://www.imgt.org/, and annotated using the Kabat numbering system (https://www.bioinf.org.uk/abs/).

Example 3. Expression of HTT proteins encoded by exon 1

Methods

For the expression and purification of recombinant huntingtin proteins encoded by exon 1 GSTHttExon1Q21 (GST-HD21), GST-HttExon1-Q35 (GSTHD35) and GST-HttExon1-Q49 (GST-HD49), the expression vector pGEX-6P-1 (GE Healthcare) encoding human huntingtin exon 1 with polyglutamine tracts of 21, 35, or 49 CAG repeats, respectively (cf. Fig. 2A), linked using a PreScission cleavage site with an N-terminal glutathione-transferase (GST) tag, was expressed in E. coH cells strain BL21. Bacterial cultures incubated overnight (37°C, 220 rpm) were diluted 1:25 and induced expression at OD600 0.5-0.6 for 4 hours by adding 1 mM IPTG (Sigma I1284) and additional incubation at 36 °C, 220 rpm. Cultures were grown in LB medium containing 100 μg/ml ampicillin at 37°C for overnight incubated cultures supplemented with an additional 1% glucose. Recombinant GST-HttExon1 proteins were purified by coupling to glutathione agarose (Sigma G4510). Briefly, the bacterial pellet was resuspended in 2040 ml of cold buffer 1 (50 mM NaH2PO4, 5 mM Tris, 150 mM NaCl, 1 mM EDTA pH8, lysozyme to a final concentration of 5 mg/ml, complete set of protease inhibitors (Roche)), incubated for 60 min on ice, sonicated, added Triton-X100 (final concentration 0.1%) and centrifuged for 90 min at 14,000 g after incubation on ice for 5 min. Glutathione-agarose was added to the supernatant, incubated for 2 h at 4°C, pelleted by centrifugation for 10 min at 1000 g and washed 2 times with cold PBS after removing the supernatant. Elution was carried out for 5 min in 1 ml of buffer 1 with 10 mM reduced glutathione, pH 9. This step was repeated from 5 to 15 times until the protein stopped eluting. The pooled supernatants were dialyzed into buffer (50 mM Tris, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% glycerol) overnight (HOMM 10 kDa, Pierce); Aliquots were stored at -80°C.

SDS-PAGE analysis

Purified recombinant GST-HttEx1 proteins were isolated by passing through a DSNPAG gradient (NuPAGE Bis-Tris 4–12%; Invitrogen, Basel, Switzerland) followed by Coomassie brilliant blue staining or electroblotting on nitrocellulose membranes. Blots were incubated with primary antibodies Mab 5492 (Chemicon, N-terminal epitope AK1-82, proline-rich domain) or NI-302.37C12, followed by secondary HRP-conjugated goat anti-mouse IgG or secondary HRP-conjugated donkey anti-human IgG. . Blots were developed using ECL and an ImageQuant LAS 4000 detection system (GE Healthcare, Switzerland).

As shown in FIG. 2B, various recombinant GST-HttEx1 proteins were successfully expressed and purified, as shown by Coomassie staining after SDS-PAGE electrophoresis.

Example 4 Determination of Aggregation Status Using Dot Blot and Filter Delay Method

To determine the aggregation kinetics of HD21, HD35 and HD49 proteins, analysis was carried out using filter delay and dot blot methods.

- 76 046697

Accordingly, the aggregation reaction was first carried out as follows: Recombinant GST-HttExon1 proteins can be expressed and purified as a fusion protein. After the cleavage of the GST tag from the hybrid protein by the PreScission (PP) protease, the aggregation reaction of the huntingtin protein encoded by exon 1 immediately begins. Before the start of the reactions, the GST-HttExon1 proteins were centrifuged at 100,000 g for 30 min. The clarified protein solution was diluted to a protein concentration of 2 μM in cold aggregation buffer (0.05 M Tris/HCL, pH7, 0.15 M NaCl, 1 mM EDTA) and added 1 mM DTT and PreScission protease (GE Healthcare). The reaction mixture was incubated at room temperature with rotation at 300 rpm; Aggregation reactions were stopped by flash freezing at 80°C at specified time intervals. After incubation for 1, 3, 5, 7, and 24 h, aliquots of the aggregation reaction products HD21, HD35, and HD49, respectively, were snap-frozen on dry ice and stored at -80°C.

For dot blot analysis, samples were thawed on ice, diluted, and transferred to a nitrocellulose blotting membrane using a filter device that applied a vacuum in a chamber beneath the membrane. For this purpose, the membrane was equilibrated with PBS, placed in a chamber and washed with 100 μl of PBS per well. Samples were loaded and pulled completely through the membrane, followed by washing 3 times with PBS. The device was disassembled, the membrane was quickly air-dried for 15 min at room temperature, blocked for 1 h at room temperature with blocking buffer (3% BSA, 0.1% Tween 20 in PBS buffer) and incubated with polyclonal antibody HD-1 (1:10,000, kindly provided by Prof. E. Wanker, MDC, Berlin). After washing, the membrane was incubated for 1 h at RT with HRP-conjugated anti-rabbit IgG antibody, and the blots were developed using ECL and an ImageQuant LAS 4000 detection system (GE Healthcare, Switzerland).

As shown by the dot blot results shown in FIG. 2C, left, HD-1 polyclonal antibody detected HD21, HD35, and HD49 proteins regardless of aggregation status.

For filter retention assays, samples were thawed on ice, diluted in denaturing buffer (4% SDS, 100 mM DTT) and transferred through a 0.2 μm cellulose acetate membrane using a vacuum chamber: For this purpose, the membrane was equilibrated at 0 .1% SDS solution in PBS was placed on a vacuum chamber and the wells were washed with 0.1% SDS. Samples were added, filtered through a membrane using vacuum, and washed 3 times with 0.1% SDS. Then the membrane was removed from the chamber, quickly dried in air, blocked for 1 hour at RT in blocking buffer (5% milk, 0.1% Tween 20 in PBS buffer), and incubated with polyclonal antibody HD-1 (1:5000, Scherzinger et al., Cell 90 (1997), 549-558) and further processed as described above.

In the filter retention assay, the first aggregates retained on the membrane were detected after 24 h of incubation and were HD35. HD49 proteins with extension due to the polyglutamine tract form insoluble aggregates already 3 hours after cleavage of the GST tag, see Fig. 2C right (FRA).

Example 5 Characterization of exon 1-encoded huntingtin aggregates

Electron microscopy (EM) was used to verify and describe the formation of HD35 and HD49 aggregates encoded by exon 1. Briefly, the products of HD49 aggregation reactions after 1, 3, and 24 h, respectively, or HD35 samples after 24 h were analyzed using electron microscopy. The samples were adsorbed on copper grids with a carbon film under glow discharge conditions. Excess samples were removed with filter paper. The grids were stained with 2% (w/v) uranyl acetate for 1 min, and excess uranyl acetate was washed off with distilled deionized water. The grids were air dried and imaged using a Philips CM100 transmission electron microscope with an accelerating voltage of 100 kV.

EM analysis of the HD35 aggregation reaction revealed larger aggregates visible by EM, resembling protofibrillar structures, after 24 h of incubation ( Fig. 2D [E]). HD49 exhibited faster aggregation kinetics, with detectable fibrils appearing after just 1 h of incubation (Figure 2D [F]) and increasing fibril size and number as aggregation progressed over time (Figure 2D [C, D, G, H]). These observations are consistent with results obtained from filter retention assays, where aggregates larger than 0.2 μm are retained on a cellulose acetate membrane, and confirm the successful formation of exon 1-encoded huntingtin aggregates; see also example 4.

Example 6: Polyproline Domain Binding Affinity of Antibody NI-302.33C11 Using Direct ELISA and EC ₅₀ Determination

A direct ELISA assay was performed to determine the half-maximal effective concentration (EC ₅₀ ) of the recombinant human-derived anti-HTT antibody NI-302.33C11 for soluble and aggregated huntingtin proteins encoded by exon 1 with 21 or 49 polyglutamine repeats. Briefly, 96-well microplates (Corning) were coated with GST-HD21, GST-HD49, or aggregated HD21 or HD49 at a concentration of 5 μg/ml in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.42). Nonspecific binding sites were blocked for 1 hour at RT solution

- 77 046697 rum PBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland). Primary antibodies were diluted to specified concentrations and incubated for 1 h at RT. Binding was determined using either HRP-conjugated FcY-specific donkey anti-human IgG or HRP-conjugated (H+L)-specific goat anti-mouse IgG, followed by measurement of HRP activity in a standard colorimetric assay. _EC50 values were then calculated using nonlinear regression using GraphPad Prism software (San Diego, USA).

EC50 values of the human-derived anti-HTT antibody NI-302.33C11 for aggregated and soluble Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats were determined using direct ELISA when coated with various formulations at a concentration of 5 μg/ml. As shown in FIG. 3A and B, antibody NI-302.33C11 bound with similar high affinity to all four species, including pathological aggregated Htt encoded by HD49 exon 1, with EC50 values of approximately 100 pM.

Example 7. Evaluation of the binding selectivity of anti-HTT antibodies using dot blot and filter delay analysis

Filter delay and dot blot analysis were performed to characterize the binding of recombinant human-derived anti-HTT antibody NI302.33C11 to soluble and aggregated huntingtin proteins encoded by exon 1 with 21, 35, or 49 polyglutamine repeats. For this purpose, aliquots of the aggregation reaction products HD21, HD35 and HD49 as described in example 4 were removed after 1, 3, 5, 7 and 24 hours of incubation, flash frozen on dry ice and stored at -80°C; dot blotting was performed as described in Example 4. The filter delay assay was also performed as described in Example 4, except that the membrane was incubated with NI-302.33C11 (1 μg/ml).

It can be noted that in the dot blot analysis (Fig. 3C, left), the NI-302.33d1 antibody preferentially detects huntingtin proteins extended by polyglutamine (poly-Q) tracts (HD49>>HD35>HD21). In addition, the signal intensity increased with increasing incubation time for the products of the HD35 and HD49 aggregation reactions.

The same was true for the results obtained in the filter retention assay (Fig. 3C, right), which showed that NI-302.33d1 detected HD35 and HD49 aggregates retained on the 0.2 μm pore size membrane. These results, based on spot protein formulations, suggest that antibody NI-302.33d1 preferentially binds aggregated HTT conformers with pathogenic polyglutamine extensions.

Example 8. Binding specificity and selectivity of anti-HTT antibodies towards irrelevant aggregating protein targets using direct ELISA

To determine the binding specificity of the recombinant antibody NI-302.33C11, which binds to the polyproline region of HTT and does not bind to irrelevant aggregating protein targets, direct ELISA was performed in 96-well microplates (Corning) coated with different target proteins at a concentration of 1-10 μg /ml in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.42). Nonspecific binding sites were blocked for 1 h at RT with PBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland). Antibody NI302.33C11 was diluted to specified concentrations and incubated for 1 h at RT. Binding was determined using an HRP-conjugated donkey anti-human Fcy IgG antibody followed by measurement of HRP activity in a standard colorimetric assay. Signals for the target protein were calculated as fold increases relative to the median signal.

It may be noted that the human-derived antibody NI-302.33C11 specifically binds HTT, namely aggregated HD49, in the absence of binding other irrelevant protein targets, including known amyloid-forming proteins, see FIG. 16A.

Example 9 Determination of the binding epitope of the anti-HTT antibody NI-302.33C11

To map the huntingtin epitope (HTT) recognized by the human-derived antibody NI-302.33C11, epitope mapping was performed using a peptide scanning assay with synthetic peptides. Briefly, peptide scanning of overlapping peptides was used for epitope mapping. The sequence of HTT, encoded by human exon 1, was synthesized as 16 linear 15-mer peptides with 10 amino acid stretches overlapping individual peptides (JPT Peptide Technologies, Berlin, Germany) and applied to nitrocellulose membranes. The membrane was activated for 5 minutes in methanol and then washed at RT in TBS for 10 minutes. Nonspecific binding sites were blocked for 2 h at room temperature in a Roti®-Block (Carl Roth GmbH+Co. KG, Karlsruhe, Germany). Human antibody NI-302.33C11 (1 μg/ml) was incubated for 3 h at RT in Roti®-Block. Primary antibody binding was determined using an HRP-conjugated donkey anti-human IgGy secondary antibody. Blots were developed using ECL and an ImageQuant LAS 4000 detection system (GE Healthcare, Switzerland).

- 78 046697

As shown in FIG. 4, pronounced binding of NI-302.33C11 to peptides No. 7, No. 8, No. 9, No. 13 and No. 14 was observed, which indicates the localization of the epitope recognized by this antibody in the huntingtin domain with polyproline repeats. The binding epitope of antibody NI-302.33C11 is accordingly predicted to be located within the amino acids HTT 35-PPRRRRRR-42 (SEQ ID No:

139) and amino acids 63-PPRRRRRRRRRR-72 (SEQ ID No: 162).

Example 10: Epitope Mapping by Binding Anti-HTT Antibody NI-302.33W1 to Various Exon 1 Encoded Peptides Using Direct ELISA

To determine the half-maximal effective concentration (EC50) of the recombinant human-derived anti-HTT antibody NI-302.33C11 for BSA-conjugated peptide fragments of huntingtin encoded by exon 1, a direct ELISA was performed with BSA-conjugated peptides of the Htt domains encoded by exon 1.

Briefly, 96-well microplates (Corning) were coated with BSA-conjugated synthetic peptides (Schafer-N, Denmark) containing N-terminal amino acids 1-19 (MATLEKLMKAFESLKSFQQ, SEQ ID No: 93), proline-rich domain sequence (PPQLPQPPPQAQPLLPQPQPP, SEQ ID No: 94), polyproline repeat sequence (PPRRRRRRRR, SEQ ID No: 95) or 14 C-terminal amino acids (PPGPAVAEEPLHRP, SEQ ID No: 96); or full-length GST-HD49Exon1 proteins at a concentration of 5 μg/ml in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO3, pH 9.42). Nonspecific binding sites were blocked for 1 hour at RT with PBS/0.1% Tween®-20 containing 2% BSA (Sigma-Aldrich, Buchs, Switzerland). Primary antibodies were diluted to specified concentrations and incubated for 1 h at RT. Binding was determined using an FcY-specific donkey anti-human IgG antibody conjugated to HRP, followed by measurement of HRP activity in a standard colorimetric assay; EC ₅₀ values were calculated using nonlinear regression using GraphPad Prism software (San Diego, USA).

As shown in FIG. 5, NI-302.33C11 bound with high affinity to BSA-conjugated polyproline peptides as well as full-length GST-HD49 with equivalent EC5o values of 30 pM. These results confirm the results of epitope mapping to the polyproline sequence according to example 9.

Example 11. Evaluation of the purity and integrity of the recombinant human antibody NI-302.33C11 to the polyproline domain

To assess the purity and integrity of the recombinant human anti-polyproline domain antibody NI-302.33C11, the primary human anti-polyproline domain antibody NI-302.33C11 was expressed by transient transfection of CHO-S cells and affinity purified with protein A in the Akta system. After desalting on a PD-10 column, the antibody was diluted in PBS. DSNPAAG analysis was then performed by obtaining 2 and 10 μg of purified recombinant human antibody NI302.33C11 to the polyproline domain under reducing conditions in a DSNPAAG concentration gradient (NuPAGE 4-12% Bis-Tris gel; Invitrogen) followed by Coomassie staining (SimplyBlue SafeStain , Invitrogen).

SDS-PAGE analysis of the recombinant human primary antibody NI-302 against the polyproline domain under reducing conditions reveals two major bands corresponding to the heavy and light chains of the antibody of the expected size, as shown in FIG. 6, and no significant contaminants or proteolytic degradation products were detected.

Example 12 Characterization of the anti-HTT antibody NI-302.33C11 in transgenic mice expressing human HTT

An immunohistochemical study was performed to evaluate the binding of the NI-302.33C11 antibody to pathological huntingtin in the brain tissues of human HTT transgenic mice. The B6.Cg-Tg(HDexon1)61Gpb/J transgenic mouse line (Mangiarini et al., Cell 87 (1996), 493-506) is a well-characterized mouse model of Huntington's disease (HD). Beginning at approximately 9 weeks of age, animals in this model develop a progressive pathology characterized by intranuclear huntingtin inclusions, which resembles Huntington's disease in humans (Naver et al., Neuroscience 122 (2003), 1049-1057). The brain hemispheres of the indicated transgenic B6.Cg-Tg(HDexon1)61Gpb/J mice at the progressive stage of the disease (270 days) were fixed in a phosphate-buffered 4% paraformaldehyde solution, embedded in paraffin, and sections 5 μm thick were made. After pretreatment with formic acid and citrate buffer, sections were incubated with 1, 5, or 50 nM human anti-HTT antibody NI-302.33C11, followed by incubation with biotinylated donkey anti-human secondary antibody (Jackson Immunoresearch; 1:250). The antibody signal was amplified using the Vectastain ABC kit (Vector Laboratories) and detected using diaminobenzidine (Pierce).

As shown in FIG. 27, the human-derived antibody NI-302.33C11 to the polyproline domain provided very pronounced staining of pathological intranuclear inclusions of neurons even at a minimal concentration of 1 nM (Fig. 27 [E-H]), which is consistent with

- 79 046697 high binding affinity of huntingtin aggregates as assessed using ELISA and dot blot. At a concentration of 5 nM or greater, the antibody additionally completely stained medium spiny neurons and provided more generalized diffuse staining also detectable in brain sections of non-transgenic animals. The presence of some degree of cross-reactivity cannot be excluded, since the polyproline epitope targeted by NI-302.33C11 was also present on numerous irrelevant proteins (Fig. 27 [F-H]).

Example 13: Determination of binding affinity and selectivity of antibody to the proline-rich domain of NI-302.63F3 using direct ELISA, and determination of EC ₅₀

To determine the half-maximal effective concentration (EC50) of the recombinant human-derived anti-HTT antibody NI-302.63F3 for soluble and aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats, a direct ELISA was performed and the EC50 was determined as described in Example 6 , higher.

It can be noted that NI-302.63F3 binds with similar high affinity to all four species, including the aggregated Htt encoded by HD49 exon 1, with EC50 values ranging from approximately 200 to 400 pM (Fig. 7A and B). Accordingly, the human HTT-derived antibody to the proline-rich domain NI-302.63F3 targets an epitope exposed on the aggregated as well as the non-truncated, more linear structured HTT protein encoded by exon 1 with high affinity in the subnanomolar range.

In addition, to determine the binding of recombinant human-derived anti-HTT antibody NI-302.63F3 to soluble and aggregated Htt proteins encoded by exon 1 with 21, 35, or 49 polyglutamine repeats, filter delay and dot blot analysis were performed as described in Example 7 above, with minor modification: incubation was carried out with 1 μg/ml antibody NI-302.63F3.

In the dot blot analysis, antibody NI-302.63F3 most strongly detected the polyglutamine-extended HD49 protein (Fig. 7C, left panel). In the filter delay assay, NI-302.63F3 detected HD35 and HD49 aggregates remaining on the 0.2 μm pore size membrane (Figure 7C, right panel). These results, based on spot protein compositions, demonstrate that the NI-302.63F3 antibody recognizes aggregated HTT conformers with pathogenic polyglutamine extensions.

In addition, to determine the binding of recombinant antibody NI-302.63F3 to irrelevant aggregating protein targets, a direct ELISA was performed as described in Example 8 above. As shown in FIG. 16B, human-derived NI-302.63F3 specifically bound to HTT, whereas no binding to irrelevant proteins was observed.

Example 14 Determination of the binding epitope of the anti-HTT antibody NI-302.63F3

To map the huntingtin epitope recognized by the human-derived antibody NI302.63F3, epitope mapping was performed on synthetic peptides as described above in Example 9.

As shown in FIG. 8, there was a pronounced binding of NI-302.63F3 to peptides No. 10 and No. 11, and a weak signal for peptides No. 8 and No. 9, which indicates the localization of the epitope recognized by this antibody in the proline-rich domain (between regions with polyproline repeats) NTT. Accordingly, the binding epitope for NI-302.63F3 is predicted to be located at the amino acid sequence HTT 43-(PPPQL)PQPPPQAQPL-57 (SEQ ID Nos: 161 and 140).

Example 15: Epitope mapping using direct ELISA for binding to various peptides encoded by exon 1 of the anti-HTT antibody NI-302.63F3

To determine the half-maximal effective concentration (EC50) of the recombinant human-derived anti-HTT antibody NI-302.63F3 for the BSA-conjugated exon 1-encoded Htt peptide fragments, a direct ELISA assay was performed with the BSA-conjugated exon 1-encoded Htt peptide domain and definition of EU ₅₀ as described in example 10.

As shown in FIG. 9, NI-302.63F3 binds with high affinity to the BSA-conjugated proline-rich peptide domain as well as full-length GST-HD49 with similar EC ₅₀ values of 200 to 300 pM. This confirms the sequence localization of the proline-rich domain determined by epitope mapping, as shown in Example 14.

Example 16. Evaluation of the purity and integrity of the recombinant human antibody NI-302.63F3 to the proline-rich domain

To assess the purity and integrity of the recombinant human antibody NI-302.63F3 to the proline-rich domain, SDS-PAGE analysis was performed as described in Example 11 above.

SDS-PAGE analysis of the recombinant human antibody NI-302.63F3 to the proline-rich domain under reducing conditions revealed two major bands corresponding to the heavy and light chains of the antibody of the expected size. No significant contaminants or products were detected

- 80 046697 proteolytic degradation, as seen in FIG. 10.

Example 17 Characterization of the anti-HTT antibody NI-302.63F3 in transgenic mice expressing human HTT

Binding of the anti-proline-rich domain antibody NI-302.63F3 to pathological HTT in human HTT-containing transgenic mouse brain tissue was assessed as described in Example 12 above, except that sections were incubated with 1 or 50 nM anti-proline-rich domain antibody.

As shown in FIG. 28 [E-F], the human-derived antibody NI-302.63F3 to the proline-rich domain revealed pronounced, highly specific staining of pathological intranuclear inclusions of neurons using concentrations of 1 and 50 nM in the striatum and cortex of R6/1 transgenic animals, which is consistent with high binding affinity to HTT aggregates as assessed using ELISA and dot blot analysis. However, as shown in FIG. 28[F], at a concentration of 50 nM, the NI-302.63F3 antibody, in addition, completely weakly stained the nucleus of medium spiny neurons.

Example 18. Determination of binding affinity and selectivity of antibodies NI-302.35W and NI302.72F10 to the C-terminal domain using direct ELISA and EC ₅₀

To determine the half-maximal effective concentration (EC ₅₀ ) of recombinant human-derived antibodies to Htt NI-302.35C1 and NI-302.72F10 for soluble and aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats, a direct ELISA was performed and determination of EC50 as described in example 6 above.

It can be noted that NI-302.35C1 binds with high affinity to all four species, including aggregated HTT encoded by HD49 exon 1, with an EC50 of approximately 2.7 nM; see fig. 11A and B. Similarly, NI-302.72F10 binds to all four species, but with a different affinity from NI-302.35C1 (aggregated HD21>>GST-HD21>aggregated HD49>>GST-HD49) (Fig. 31C) which may be explained by the presence of different epitopes recognized by both antibodies (Fig. 20).

Accordingly, the human-derived HTT antibodies NI-302.35C1 and NI-302.72F10 against the C-terminal domain target an epitope exposed on aggregated as well as soluble forms of HTT with affinities in the low nanomolar range.

In addition, filter delay and dot blot analysis were performed to characterize the binding of recombinant human-derived anti-HTT antibodies NI302.35C1 and NI-302.72F10 to soluble and aggregated Htt proteins encoded by exon 1 with 21, 35, or 49 polyglutamine repeats. as described in example 7 above.

In dot blot analysis, antibody NI-302.35C1 predominantly detected constructs with HTT extended by polyglutamine (poly-Q) tracts (HD49>HD35>>HD21, Fig. 11C, left). In addition, the signal intensity increases with increasing incubation time for the products of the HD35 and HD49 aggregation reactions. Similarly, the NI-302.72F10 antibody detected constructs with HTT extended by polyglutamine (poly-Q) tracts, although with different preferences (HD35>>HD49>HD21), and the signal intensity increased only with increasing incubation time of the reaction products HD35 aggregation (Fig. 32C).

In the filter delay assay, NI-302.35C1 detected HD35 and HD49 aggregates retained on the 0.2 μm pore size membrane (Figure 11C, right), while NI-302.72F10 detected only HD35 aggregates (Figure 32C, right ). These results, based on membrane-bound protein compositions, suggest that antibodies NI-302.35C1 and NI-302.72F10 preferentially target aggregated HTT conformers with pathogenic poly-Q extensions.

In addition, to determine the binding of recombinant antibodies NI-302.35C1 and NI-302.72F10 to irrelevant aggregating protein targets, a direct ELISA was performed as described in Example 8 above. As shown in FIG. 16C, derived from human NI-302.35C1, and also shown in FIG. 33C human-derived NI-302.72F10 specifically bound to HTT, while no binding to irrelevant proteins was observed.

Example 19 Determination of the binding epitope of the anti-HTT antibody NI-302.35C1

To map the huntingtin epitope recognized by the human-derived antibody NI302.35C1, epitope mapping was performed on synthetic peptides as described above in Example 9.

Determination of the binding epitope of antibody NI-302.35C1 by scanning overlapping peptides did not result in a specific signal. Accordingly, treatment of this antibody in a manner identical to other NI-302 anti-HTT antibodies did not result in any specific signal from the individual peptides for unknown reasons. This epitope was successfully mapped to the C-terminal peptide by conjugating the latter with BSA, see also example 20.

Example 20 Epitope Mapping by Direct ELISA Linkage

- 81 046697 with different peptides of the C-terminal domain encoded by exon 1, antibodies to HTT NI-302.35C1

To determine the half-maximal effective concentration ( _EC50 ) of the recombinant human-derived anti-HTT antibody NI-302.35C1 for BSA-conjugated Htt exon 1-encoded peptide fragments, a direct ELISA was performed with BSA-conjugated exon 1-encoded Htt domain peptides. and carried out the determination of EC50 as described in example 10.

As shown in FIG. 12, NI-302.35C1 binds with high affinity to the BSA-conjugated C-terminal peptide as well as full-length GST-HD49 with an EC50 value of approximately 0.7 nM and 3.2 nM, respectively. These results correspond to the localization of the epitope in the sequence of the C-terminal region of HTT encoded by exon 1 (71-PPGPAVAEEPLHRP-85, SEQ ID No: 96). When the same C-terminal peptide was directly coated on the plate, only minor binding was observed (EC ₅₀ >100 nM, data not shown), suggesting that presentation of the BSA-conjugated peptide enhances binding to the epitope; this may explain the fact that the epitope mapping method of peptide scanning according to example 20 did not give results.

Example 21. Evaluation of the purity and integrity of the recombinant human antibody NI-302.35C1 to the proline-rich domain

To assess the purity and integrity of the recombinant human antibody to the C-terminal domain of NI302.35C1, SDS-PAGE analysis was performed as described in Example 11 above.

SDS-PAGE analysis under reducing conditions of the recombinant human antibody NI302.35C1 to the C-terminal domain revealed two main bands corresponding to the heavy and light chains of the antibody of the expected size, while no significant contaminants or proteolytic degradation products were detected (Fig. 13) .

Example 22. Characterization of the anti-HTT antibody NI-302.35C1 in human HTT transgenic mice

The binding of the NI-302.35C1 antibody to the C-terminal domain of pathological human HTT in brain tissues of transgenic mice was assessed as described in Example 12 above, with the difference that the sections were incubated with 5 or 50 nM of the antibody to the C-terminal domain.

As shown in FIG. 29 [E-F], a human-derived antibody to the C-terminal domain of NI302.35C1 at concentrations of 5 and 50 nM provided pronounced staining of pathological intranuclear inclusions of neurons in the striatum of R6/1 transgenic animals, which is consistent with high binding affinity to HTT aggregates assessment using ELISA and dot blotting.

Example 23: Evaluation of the Effects of Human-Derived Antibodies Targeting HTT on Spine Density in Hippocampal Slice Cultures

Antibody expression and purification

Human-derived antibodies targeting individual Htt domains encoded by exon 1 were expressed by transient transfection of CHO-S cells and affinity purified with protein A in the Akta system. After desalting on a PD-10 column, antibodies were diluted in PBS. Endotoxin levels less than <10 U/mL were confirmed.

Hippocampal slice culture

Organotypic hippocampal slice cultures were prepared and cultured as described in Stoppini et al., J Neurosci Methods. (1991) 37(2): 173-82. Briefly, 6-8 day old B6CBA-Tg(HDexon1)62Gpb/1J transgenic and non-transgenic littermates were decapitated, brains were removed, both hippocampi were isolated, and sections were cut at 400 μm thickness. This method provides thin sections with a thickness of 1-4 cell layers, which are characterized by a well-preserved organotypic organization. Sections were cultured on Millicell plate culture inserts (0.4 μm, Millipore) in six-well plates containing 1 ml of culture medium (46% HEPES-modified Eagle's minimal essential medium, 25% modified Eagle's basal medium, 25% heat-inactivated horse serum , 2 mM glutamine, 0.6% glucose, pH 7.2). Culture plates were kept at 37°C in a humidified atmosphere containing 5% CO2. The slices were cultured for 7 days before experiments. The culture medium was replaced every 2-3 days. On day 7, antibodies were added at a concentration of 10 μg/ml. On day 10, in vitro slice cultures were infected with Sindbis virus using the droplet method (Shahani et al., J Neurosci. 31 (2006), 6103-6114). For spine analysis, cultures were fixed at day 4 postinfection (14 days in vitro). The sections were left attached to the membrane of the culture plate to preserve the structure of the hippocampus and washed with PBS. Then the sections were fixed in a 4% solution of paraformaldehyde in PBS containing 4% sucrose for 2 hours at 4°C. Each dendrite was photographed and spines were analyzed in a 30-45 µm long section. For each antibody treatment, quantitation was performed on 6-13 sections for a group of 12 transgenic animals. Data shown are mean ± SEM. *p<0.05 (MWU), # p=0.05.

Quantification of dendritic spine density in hippocampal slice cultures.

- 82 046697 Tg(HDexon1)62Gpb/1J transgenic mice (Fig. 17 B, D) revealed a significant reduction in density of 53% relative to non-transgenic littermates (Fig. 17 A, C, E), using hippocampal slices from 6-day-old animals after 14 days in vitro under the experimental design described above. When human-derived antibody NI-302 was added at a concentration of 10 μg/ml for seven days, there was a significant reduction in spine density loss with NI-302.31F11 antibody (Figure 17F, p < 0.05, t test) and NI-302.63F3 (p = 0.05, t test), while antibodies NI-302.33C11 and NI-302.35W did not provide a clear effect under the conditions tested.

The significant reduction in spine density compared to non-transgenic littermates observed in hippocampal slice cultures from Tg(HDexon1)62Gpb/1J transgenic mice suggested that this model is suitable for testing in vitro the activity of candidate anti-HTT antibodies in interacting with toxic HTTs. In this model, antibodies NI-302.63F3 and NI-302.11F11, both targeting the P-rich domain of Htt exon 1, were able to increase spine density compared with the isotype control antibody. This indicates that these antibodies are able to mitigate the toxic effects on spine density caused by the expression of pathological HTT with polyglutamine extensions.

Example 24 Penetration of NI-302 Antibodies into the Brain in the R6/1 Animal Model

To test the penetration of antibodies to HTT according to the present invention, a transgenic mouse model was used. Specifically, Tg(HDexon1)61Gpb/J transgenic mice carry a 1.9 kb transgene isolated from a phage containing a genomic clone derived from a patient with Huntington's disease (HD) and including the 5' end human huntingtin gene (HTT). It consists of approximately 1 kb. sequences of the 5' untranslated region, exon 1 (with extensions from CAG repeats, ~130 units) and the first 262 bp. intron 1. These constructs were microinjected into CBAxC57BL/6 one-cell embryos. The R6 founder male was crossed with CBAxC57BL/6 females to produce multiple founder lines. In the R6/1 founder line mice, the transgene is integrated as a single intact universally expressed copy. Transgenic mice with a mixed CBAxC57BL/6 genotype background were backcrossed with C57BL/6J for over 12 generations to generate the congenic line B6.Cg-Tg(HDexon1)61Gpb/J.

R6/1 transgenic mice exhibit a progressive neurological phenotype reproducing many of the hallmarks of HD, including choreiform movements, involuntary stereotypic movements, tremors and epileptic seizures, as well as components of non-motor disorders, including unusual vocalizations. They experience frequent urination and loss of body weight and muscle mass during the course of the disease. Neurologically, they develop neuronal intranuclear inclusions (NIIs) that contain both HTT and ubiquitin proteins. Such NNs have also been identified in human patients with HD. The reported age of onset of GD symptoms for this 6/1 line is 15 to 21 weeks.

Experimental animals that were given a classic ear tag for identification differed in the following characteristics:

Line: Hemizygous B6.Cg-Tg(HDexon1)61Gpb/J (Mangiarini et al., Cell, 87 (1996), 493-506)

Source: Jackson Laboratory, Maine, USA

Gender: Males and females

Initial age: 230-260 days

Cohorts:

NI-302.31F11 Total: 3 males

NI-302.35C1 Total: 3 males

Base Total: 3 males

To obtain homogenized spinal cords, B6.CgTg(HDexon1)61Gpb/J transgenic mice were deeply anesthetized and transcardially perfused with cold phosphate buffered saline through the left ventricle using a peristaltic pump. The brains were removed and flash frozen on dry ice. Tissue samples were homogenized 1:10 (w/v) in diethanolamine (DEA) buffer (50 mM NaCl, 0.2% DEA, complete protease inhibitor kit, Roche Diagnostics) using a hand-held ultrasonicator (Sartorius, Labsonic M ). Samples were centrifuged for 30 min at 100,000 x g at 4°C; Aliquots of the supernatant were stored at −80°C until analysis.

In the human IgG drug level sandwich ELISA described below, plasma levels of human antibodies NI-302.35C1, NI-302.11F11 were determined using the corresponding recombinant antibody at a known concentration as a standard. 96-well microplates (Corning) were coated with donkey anti-human IgG (709-005-149, Jackson Immunoresearch) at a concentration of 1 μg/ml in 50 mM carbonate coating buffer, pH 9.6. Nonspecific binding sites were blocked for 1 h at RT with PBS/0.1% Tween®-20 containing 2% BSA (Sigma, Buchs, Switzerland). Plasma samples were diluted to 1:20,000 and 1:100,000, brain homogenates

- 83 046697 brains were diluted to 1:5 and 1:50, and all samples were incubated for 1 hour at RT along with standards for dilution curves. Binding was determined using an HRP-conjugated anti-human detector antibody (709-036-098, Jackson Immunoresearch), followed by measurement of HRP activity in a standard colorimetric assay. Concentrations in plasma and spinal cord samples were calculated from individual standard curves. The values given in table. 6-1 are the averages of 2 independent ELISA experiments.

Plasma and brain samples were obtained 2 days after a single intraperitoneal injection of 50 mg/kg antibodies NI-302.31F11, NI-302.35C1 into R6/1 transgenic mice. Antibody levels in plasma and brain homogenates were determined using a sandwich ELISA assay for human IgG (Fig. 18A and B). The brain/plasma drug level ratio was determined for human-derived antibodies NI-302.31F11 and NI-302.35C1 to be 0.13±0.02%/0.21±0.06%, respectively. These results indicate that the tested NI-302 antibodies penetrated into the brain of HTT transgenic mice within 48 hours within the expected range.

Example 25: Determination of Binding Affinity and Specificity of Additional Antibodies Identified in the Present Invention

To determine the half-maximal effective concentration (EC ₅₀ ) of additional identified recombinant human-derived antibodies to Htt for soluble and aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats, a direct ELISA was performed with coating of different compositions at a concentration of 5 μg/ ml and determined the EC50 as described in example 6 above.

The determined EC50 values for different types of HTT are shown in Fig. 19, and also in FIG. 31 (A, DF) and summarized in FIG. 20. Most human-derived antibodies bound with high affinity with EC ₅₀ values in the subnanomolar or low nanomolar range. Some candidate antibodies, such as NI-302.37d2, NI-302.55D8, NI-3O2.11A4, NI-302.22H9, or NI-302.64E5, appear to preferentially bind to the non-truncated GST-HTT protein, other antibodies such as NI-302.74C11, NI-302.71F6, NI-302.4A6, NI-302.12H2 or NI-302-8M1 demonstrated high binding affinity to all HTT formulations with similar _EC50 values in the ELISA assay. NI-302.15F9 showed an approximately 5-fold preference for HD49 over HD21, with EC ₅₀ values ranging from 5 to 35 nM. Antibody 33C11 was used as a control in this experiment (FIG. 32G).

Accordingly, a panel of high-affinity recombinant human HTT-specific antibodies was cloned from memory B cells derived from cohorts of healthy elderly human donors, recombinantly expressed and characterized. In addition, to search for additional redundant antibodies, screening was initiated in a cohort of selected presymptomatic patients with Huntington's disease (HD), carriers of extensions due to CAG repeats of different lengths.

To further characterize the binding of identified recombinant human-derived HTT antibodies NI-302 to soluble and aggregated Htt proteins encoded by exon 1 with 21, 35 or 49 polyglutamine repeats, a filter delay assay was performed using a primary antibody at a concentration of 0.2 μg/ ml, and dot blot analysis using a primary antibody at a concentration of 1 μg/ml as described in Example 7 above.

As shown in FIG. 21 and 32 (A, G), in the dot blot analysis (left side of the figure, Dot blots), most of the antibodies studied showed preferential detection of HTT proteins extended by polyglutamine (poly-Q) tracts (HD49>>HD35>HD21). In addition, the signal intensity increased with increasing incubation time for the products of the HD35 and HD49 aggregation reactions, in particular for antibodies NI-302.15F9, NI-302.71F6 (Fig. 21, first row of blots) and NI-30264E5 (Fig. 32 A, left). In filter delay analysis, NI-302.15F9, NI-302.71F6 (Fig. 21, right, FRA) and NI-302-64E5 (Fig. 32 A, right) detected SDS-stable HD35 and HD49 aggregates that were retained at membrane with a pore size of 0.2 μm pore size, while other antibodies, such as NI-302.44D7 and NI-302.37C12 (Fig. 21, second row of blots), or NI-302.4A6, NI-302.12H2 and NI- 302.8M1 (Fig. 32 D, E, F) did not bind to the aggregates on the filter. In this experiment, antibody 33C11 was used as a control (Fig. 32 G). These results, based on spot protein compositions, suggest that some of the cloned NI-302 antibodies exhibit a preference for aggregated HTT conformers with pathogenic polyglutamine extensions.

In addition, the binding specificity of the identified antibodies to irrelevant proteins, in particular aggregate-forming proteins, was assessed using a direct ELISA (Fig. 22 and Fig. 33 A, D-F) as described in Example 8 above. The results showed that the majority of human-derived NI-302 antibodies tested specifically bound to HTT, while there was no binding to other irrelevant proteins tested.

Example 26. Binding assessment of human-derived HTT antibodies and mapping of their epitopes

- 84 046697

To map the HTT epitope recognized by the newly identified human-derived antibodies, epitope mapping was performed on synthetic peptides as described above in Example 9 (FIGS. 23 and 35 A, DF). In addition, the half-maximal effective concentration (EC ₅₀ ) of anti-HTT antibodies for BSA-conjugated Htt peptide fragments encoded by exon 1 was determined using direct ELISA with BSA-conjugated peptides of Htt domains encoded by exon 1, and the EC50 was determined according to described in example 10.

Example 27: Evaluation of HTT Antibody Binding in Transgenic Mice Expressing Human HTT

Determination of the characteristics of the binding of identified antibodies to pathological human NTTs in the brain tissues of transgenic mice was assessed as described in example 12 above, with the difference that the sections were incubated with antibodies to HTTs at concentrations of 5 nM (74C11, 39C12, 11A4, 22H9, 78H12 , 37C12, 7D8, 72F10) or 50 nM (15F9, 71F6, 55D8, 44D7, 7A8, 64E5). As shown in FIG. 24, identified human-derived anti-HTT antibodies provided striking, highly specific staining of pathological intranuclear inclusion neurons in the striatum and cortex of R6/1 transgenic animals, also demonstrated for antibodies NI-302.33C11, NI-302.63F3 and NI-302.35C1, as described above . These results are consistent with the high binding affinity of the HTT aggregates as assessed by ELISA and dot blot in Example 26.

Example 28 Determination of the main characteristics of the R6/1 transgenic mouse model of Huntington's disease (HD)

Tg(HDexon1)61Gpb/J transgenic mice carry a 1.9-kb transgene isolated from a phage containing a genomic clone from a HD patient and including the 5' end of the human huntingtin (HTT) gene. It consists of approximately 1 kb. sequences of the 5' untranslated region, exon 1 (with extensions from CAG repeats, ~130 units) and the first 262 bp. intron 1. The indicated constructs were microinjected into CBAxC57BL/6 one-cell embryos. The R6 founder male was crossed with CBAxC57BL/6 females to produce several founder lines (Mangiarini et al., Cell 87 (1996), 493-506). In the R6/1 founder line mice, the transgene is integrated as a single intact universally expressed copy. Transgenic mice with a mixed CBAxC57BL/6 genotype background were backcrossed with C57BL/6J for over 12 generations to generate the congenic line B6.Cg-Tg(HDexon1)61Gpb/J. R6/1 transgenic mice exhibit a progressive neurological phenotype reproducing many of the hallmarks of HD, including choreiform movements, involuntary stereotypic movements, tremors and epileptic seizures, as well as components of non-motor disorders, including unusual vocalizations. They experience frequent urination and loss of body weight and muscle mass during the course of the disease. Neurologically, they develop neuronal intranuclear inclusions (NIIs) that contain both HTT and ubiquitin proteins. Such NIIs have also been identified in human patients with HD. The reported age of onset of HD symptoms for this 6/1 strain ranges from 15 weeks to 21 weeks (Naver et al., Neuroscience 122 (2003), 1049-1057; Hodges et al., Genes Brain Behav. 7(3) (2008) , 288-299).

R6/1 transgenic mice, obtained from Jackson Laboratories, were bred and longitudinally studied for behavioral phenotype, long-term body weight dynamics, total brain weight, histopathological parameters and survival, according to FIG. 25. Overall, the results are consistent with published data and indicate that this mouse strain is suitable for use as a preclinical model to study the efficacy of the human-derived antibody NI-302 targeting aggregated HTT.

Example 29. Determination of the main characteristics of the HD model using transgenic mice N171-82Q

The B6C3-Tg(HD82Gln)81Dbo/J (N171-82Q) transgenic mouse line (Schilling et al., Hum Mol Genet. 8(3) (1999), 397-407) is a well-characterized mouse model of HD. Transgenic B6C3-Tg(HD82Gln)81Dbo/J (N171-82Q) mice express an N-terminally truncated human HTT cDNA that encodes 82 glutamine residues and spans the first 171 amino acids. The modified HTT cDNA is under the control of the mouse prion protein promoter. Expression is observed in neurons of the central nervous system. Mice expressing this transgene are apparently healthy at birth and during the first 1-2 months of life. However, these mice do not gain body weight; they develop tremors, hypokinesis, and impaired coordination. They have an abnormal gait and frequent tucking of the hind limbs. Life expectancy is 5-6 months. Studies using HTT antibodies show diffuse nuclear labeling and numerous immunoreactive nuclear inclusions in several neuronal populations. In addition, obvious neurite damage was observed.

N171-82Q transgenic mice obtained from Jackson Laboratories were bred and longitudinally studied for behavioral phenotype, long-term body weight dynamics, end-stage total brain weight, histopathological scores, and survival (FIG. 26). Overall, the results obtained are consistent with published data and indicate that this mouse strain, in addition to the mouse strain described in Example 29, is suitable for use as a

- 85 046697 preclinical model to study the effectiveness of the human-derived antibody NI-302 targeting aggregated HTT.

Example 30. Evaluation of Neuronal Inclusion Staining in Patients with Huntington's Disease (HD)

To evaluate staining of neuronal inclusions with the identified antibodies of the present invention, immunohistochemical analysis was performed in patients. Binding of identified antibodies to pathological HTT in human brain tissue was assessed as described in Example 12 above, with the difference that incubation of sections was performed using concentrations of 50 nM for antibody NI-302.33C11, 50 nM for antibody NI-302.63F3 or 100 nM for antibody NI-302.35C1.

As shown in FIG. 27, immunohistochemical analysis with the polyproline region binding antibody NI-302.33C11 showed staining of intranuclear inclusion neurons in cortical neurons of patients with Huntington's disease (Fig. 27 A-D) at a concentration of 50 nM, and in striatal neurons of transgenic animals B6.Cg-Tg(HDexon1) 61Gpb/J) 270 days old in late stage disease at 1 nM (E) and 5 nM (F), while no staining was detected in non-transgenic littermates (G) unless staining uses a primary antibody (H) or if control tissue samples not affected by Huntington's disease were stained with NI-302.33C11 at a concentration of 50 nM. Antibodies NI-302.63F3 to the proline-rich domain (Fig. 28) and NI-302.35C1 to the C-terminal domain (Fig. 29) in immunohistochemical analysis at concentrations of 50 nM for NI302.63F3 or 100 nM for NI-302.35C1 showed staining of intranuclear inclusions neurons (A-C) and staining of some neurites (D) of cortical neurons in four different patients with Huntington's disease (A-D). Staining can also be observed in striatal neurons of 270-day-old B6.Cg-Tg(HDexon1)61 Gpb/J) transgenic animals in the late stage of the disease using concentrations of 1 nM (E) and 50 nM (F). No staining was detected in non-transgenic littermates (G) if the primary antibody (H) was not used in the staining, or if control tissue samples not affected by Huntington's disease were stained with 50 nM NI-302.63F3 or NI-302.35C1 at 50 nM. concentrations of 100 nM, respectively.

In contrast to the specific anti-HTT antibodies of the present invention, additional tissue staining was observed when immunohistochemical analysis was performed with the commercially available poly-Q antibody Mab1574 (1:2000, Chemicon), i.e. more general staining of nuclei and cytoplasm and staining of some neurites (Fig. 30 A, D) of cortical neurons in four different patients with Huntington's disease and in striatal neurons of presymptomatic transgenic animals B6.CgTg(HDexon1)61Gpb/J) at a late stage of the disease at age 150 days (Fig. 30 E) and 270 days (Fig. 30 F).

Example 31: Determination of binding affinity and selectivity of the antibody NI-302.7D8 to the polyQ/poly-P domain using direct ELISA, and determination of EC ₅₀

To determine the half-maximal effective concentration (EC ₅₀ ) of the recombinant human-derived anti-HTT antibody NI-302.7D8 for soluble and aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats, a direct ELISA was performed and the EC 50 was determined as described in the example 6, above.

It may be noted that NI-302.7D8 binds with similar high affinity to soluble GST-HD21 and aggregated HD21 with _an EC50 of approximately 50 to 100 nM, but shows a preference for the extended, more pathogenic form of aggregated HD49 and soluble GST-HD49. The EC ₅₀ for which is 17 and 6 nM, respectively (Fig. 31B).

Accordingly, the human-derived antibody NI-302.7D8 to the HTT poly^/poly-P domain targets an epitope exposed on the aggregated as well as the non-truncated, more linear HTT protein encoded by exon 1 with high affinity in the low nanomolar range .

In addition, to determine the binding of recombinant human-derived anti-HTT antibody NI-302.7D8 to soluble and aggregated Htt proteins encoded by exon 1 with 21, 35, or 49 polyglutamine repeats, a 1 μg/mL filter assay was performed using the filter delay assay. and dot blotting as described in Example 7 above.

It may be noted that in the dot blot assay (FIG. 32B, left panel), antibody NI302.7D8 was equally effective in detecting huntingtin proteins extended by polyglutamine (poly-Q) tracts (HD49=HD35=HD21). In the filter delay assay (FIG. 32B, right panel, FRA), antibody NI-302.7D8 did not bind to the SDS-stable HD21, HD35, or HD49 aggregates on the filter membrane.

In addition, to determine the binding of recombinant antibody NI-302.7D8 to irrelevant aggregating protein targets, a direct ELISA was performed as described in Example 8 above. As shown in FIG. 33B, human-derived antibody NI-302.7D8 specifically bound to HTT, while no binding to irrelevant proteins was observed.

Example 32. Determination of the binding epitope of antibodies to HTT NI-302-64E5, NI-302.7D8 and NI302.72F10

To map the huntingtin epitope recognized by human-derived antibodies

- 86 046697

NI-302-64E5, NI-302.7D8 and NI-302.72F10, performed epitope mapping on synthetic peptides as described above in Example 9.

In fig. 35A shows pronounced binding of NI-302-64E5 to peptides #10 - #12, indicating that the epitope recognized by this antibody is located in the proline-rich domain of huntingtin. The NI-3O2.64E5 binding epitope is accordingly predicted to be located within the HTT amino acids 48-PQPPPQAQPL-58 (SEQ ID No: 200). As shown in FIG. 35 V, a pronounced binding of NI-302.7D8 to peptides No. 6-8 was observed, which indicates the localization of the epitope recognized by this antibody in the huntingtin domain containing polyglutamine/polyproline repeats. The binding epitope for NI-302.7D8 is accordingly predicted to be located within the HTT amino acids 28-QQQQQQQPPP-37 (SEQ ID No: 201). At the same time, pronounced binding of NI-302.72F10 to peptides No. 15 and No. 16 was observed, indicating the localization of the epitope recognized by this antibody in the N-terminal domain of HTT (Fig. 35 C). The binding epitope for NI-302.72F10 is accordingly predicted to be located within the amino acid HTT 70-PPPGPAVAEEPLH-82 (SEQ ID No: 202).

Example 32 Determination of binding affinity and selectivity of antibody to the N-terminal domain of NI-302.15E8 using direct ELISA and determination of EC ₅₀

To determine the half-maximal effective concentration (EC ₅₀ ) of the recombinant human-derived anti-HTT antibody NI-3O2.15E8 for soluble and aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats, a direct ELISA was performed and the EC ₅₀ determination was performed according to described in example 6 above.

It can be noted that NI-3O2.15E8 binds with higher affinity to non-aggregated GST-HD49 and GST-HD21, and with lower affinity to aggregated HD49 and HD21, see FIG. 14A and B. Accordingly, the human HTT antibody to the N-terminal domain of NI-3O2.15E8 targets an epitope exposed on both aggregated and soluble forms of HTT, but has greater affinity for soluble forms of HTT.

Example 33 Epitope mapping by direct ELISA coupling of various exon 1-encoded peptides to anti-HTT antibody NI-302.15E8

To determine the half-maximal effective concentration (EC50) of the recombinant human-derived anti-HTT antibody NI-3O2.15E8 for BSA-conjugated peptide fragments of huntingtin encoded by exon 1, a direct ELISA was performed with BSA-conjugated peptides of the Htt domains encoded by exon 1 and EC ₅₀ was determined as described in example 10.

As shown in FIG. 15, NI-3O2.15E8 binds with high affinity to the first 19 BSA-conjugated amino acids at the N-terminus as well as to full-length GST-HD49 with an EC ₅₀ of approximately 0.1 or 15, respectively.

Example 34. Effect of anti-HTT antibody NI-302.35C1 on behavioral deficits in human HTT transgenic mice

An elevated plus maze test was performed to measure anxiety behavior and a vertical rod test to measure locomotor activity and coordination in human HTT transgenic mice to study the anti-HTT antibody NI-302.35W in vivo.

Groups and treatment

For behavioral analysis, two groups of transgenic (tg) mice (n = 24, 12 male/12 female) B6.Cg-Tg(HDexon1)61Gpb/J as described in Example 12 and one group of wild-type (wt) mice were used. In the transgenic mouse groups, animals were treated with intraperitoneal administration of either 30 mg/kg NI-302.35C1 mouse hybrid antibody or vehicle starting at 6–7 weeks of age until mice developed an end-stage phenotype at 7–9 months of age; wild-type mice were injected with the same volumes of vehicle. The elevated plus maze and vertical rod maze behavioral tests were performed at 16 and 18 weeks of age, respectively.

Elevated Plus Maze Test

The elevated plus maze test was performed according to Naver et al., Neuroscience 122 (2003), 1049-1057. The labyrinth was raised to a height of 50 cm above the floor surface. Four arms of the labyrinth (30 cm x 5 cm) extended from the central platform, forming a cross. Two arms, located one opposite the other, were limited by walls 15 cm high (closed arms); other arms were not restricted by anything (open arms). The test was carried out at the beginning of the dark phase of the animals' cycle under illumination in open arms in the range of 40 lux. Each mouse was placed in the center of the plus maze, facing the open arm. The experiment lasted for 5 min and was recorded using a video recording system (VideoMot Software, TSE Systems). Between sessions, the maze was rinsed with water and dried with a paper towel. The number of entries into the open and closed arms was then counted, and the time spent in the open and closed compartments was also assessed. Entrance is defined as the placement of all four paws of an animal in one arm. The number of entries into open arms and the time spent in open arms are used as measures of open space-induced anxiety in mice.

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Vertical rod test

The vertical rod test is widely used to evaluate basal ganglia-related movement disorders in mice; see, for example, Matsuura et al. J. Neurosci. 73 (1997), 45-48; Sedelis et al. Behav Brain Res. 125(2001), 109-125, Fernagut et al., Neuroscience 116 (2003), 1123-1130. Briefly, animals were placed head up on top of a 50 cm long vertical wooden rod (with a 1 cm diameter). The base of the rod was placed in the home cage. When placed on the rod, the animals turn head down and descend along the rod into their home cage. On the day of testing, animals were tested three times, measuring the total time required to descend (ttotal). The results of the third daily testing are shown in FIG. 24 V.

As shown in FIG. 34A, R6/1 animals that received NI-302.35C1 spent less time in the open arms of the maze, entered the open arms less often, and made fewer unprotected head tilts in the open arm compared to base-treated R6/1 animals. Thus, R6/1 mice treated with NI-302.35C1 exhibited a phenotype characterized by greater anxiety compared to non-transgenic littermates. Moreover, as shown in FIG. 24B, NI-302.35C1-treated R6/1 animals exhibited improved performance in the vertical rod test compared to vehicle-treated R6/1 animals, reaching levels similar to those of non-transgenic animals. Overall, the NI-302.35C1 antibody of the present invention has beneficial effects on behavioral performance and performance-related tasks in transgenic mice expressing human HTT.

Example 35 Sequence Alignment of Anti-HTT Antibodies

Determination of percent identity or similarity was performed using standard parameters of the BLASTn program as described in the Definitions section of the present invention. As shown in FIG. 36, all antibodies of the present invention are rich in tyrosine residues in the CDR regions.

Example 36. Preparation of bispecific antibodies to HTT

The preparation of bispecific antibodies can be carried out as generally described by Brennan; see above. The starting material for the production of bispecific antibodies is represented by intact IgG antibodies to HTT according to the present invention, recognizing any of the polyproline region, polyglutamine/polyproline region, proline-rich region, C-terminal region or N-terminal region of the HTT protein encoded by exon 1 as described in Section Examples and general information in Fig. 20. Antibodies are treated with pepsin for three hours at 37°C after treatment with acetate buffer, pH 4.0, to cleave the Fc part of the antibody. The reaction is stopped by raising the pH to 8 by adding Tris buffer. Then an equal volume of a mixture of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) is added to this solution and incubated with thionitrobenzoate (TNB) for 20 hours at room temperature. The molar ratio of the DTNB-TNB mixture, established by incubating a 40 mM DTNB solution with a 10 mM DTT solution for several minutes, was 20:30. After additional reduction of two modified F(ab') fragments with 0.1 mM DTT for 1 hour at 25°C, the correspondingly obtained fragments F(ab')-TIII and F(ab')-SH are hybridized with bispecific F (ab') ₂ fragment for 1 hour at 25°C. Bispecific F(ab') ₂ fragments were purified by gel filtration (Superdex 200 column).

Example 37. Determination of binding affinity and selectivity of bispecific antibodies to HTT using direct ELISA and EC50

To determine the half-maximal effective concentration (EC50) of bispecific HTT antibodies with soluble and aggregated Htt proteins encoded by exon 1 with 21 or 49 polyglutamine repeats, a direct ELISA and EC ₅₀ determination are performed as described in example 6 above. Bispecific antibodies to HTT bind with high affinity to all four species, including aggregated HTT encoded by HD49 exon 1, and equally target their corresponding epitopes exposed on aggregated as well as soluble forms of HTT, with high affinity in the low nanomolar range. Additionally, to characterize the binding of HTT bispecific antibodies to soluble and aggregated Htt proteins encoded by exon 1 with 21, 35, or 49 polyglutamine repeats, filter delay and dot blot analysis were performed as described in Example 7 above. In dot blot analysis, bispecific antibodies to HTT preferentially detect HTT constructs extended by polyglutamine (poly-Q) tracts. In addition, the signal intensity increases with increasing incubation time for the products of the HD35 and HD49 aggregation reactions. In the filter delay assay, bispecific antibodies to HTT detect HD35 and HD49 aggregates remaining on a membrane with a pore size of 0.2 μm. Due to dual specificity for HTT and the previously observed binding of individual antibodies to membrane-bound protein moieties, bispecific antibodies to HTT are predicted to preferentially target aggregated conformational forms of HTT with pathogenic polyglutamine extensions. In addition, to determine the binding of bispecific anti-HTT antibodies to irrelevant aggregating protein targets,

- 88 046697 carry out direct ELISA as described in example 8 above. In this context, bispecific antibodies to HTT specifically bind to HTT, while binding to irrelevant proteins may not be observed.

Claims (26)

1. A human-derived monoclonal antibody to huntingtin (HTT) or an HTT-binding fragment of said antibody, which contains in its variable region light (VL) and heavy (VH) chain variable domains, and a constant domain, wherein said antibody or HTT-binding fragment thereof recognize an epitope in the C-terminal region of exon 1 of the HTT gene, and wherein said antibody or HTT-binding fragment of said antibody contains in its variable region: (a) Complementarity determining regions (CDRs) of the VH and _VL amino acid sequences of antibody NI-302.35C1 shown in FIG. 1C or 1AF, and presented as: (i) a V _H sequence (SEQ ID NO: 9) and (ii) a V _L sequence (SEQ ID NO: 11), wherein one or more of said CDRs may contain one or two conservative amino acid substitutions; or (b) the VH and VL amino acid sequences of antibody NI-302.35C1 according to FIG. 1C or fig. 1AF. 2. A human-derived monoclonal antibody to huntingtin (HTT) or an HTT-binding fragment of said antibody, which contains in its variable region light (VL) and heavy (VH) chain variable domains, and a constant domain, wherein said antibody or HTT-binding fragment thereof recognize an epitope in the C-terminal region of exon 1 of the HTT gene, and wherein said antibody or HTT-binding fragment of said antibody contains in its variable region: (a) Complementarity determining regions (CDRs) of the amino acid sequences V _H and V _L of antibody NI-302.72F10 shown in FIG. 1Z or 1AT, and presented as: (i) a V _H sequence (SEQ ID NO: 176 or 178) and (ii) a V _L sequence (SEQ ID NO: 180 or 182), wherein one or more of said CDRs may contain one or two conservative amino acid substitutions; or (b) the VH and VL amino acid sequences of antibody NI-302.72F10 according to FIG. 1Z or 1AT. 3. An antibody or HTT-binding fragment of said antibody according to claim 1 or 2, characterized in that said antibody or fragment is of the IgG type. 4. An antibody or an HTT-binding fragment of said antibody according to any one of claims 1 to 3, characterized in that said light chain is a kappa chain (k). 5. An antibody or HTT-binding fragment of said antibody according to any one of claims 1-4, which is capable of binding aggregated forms encoded by exon 1 of HTT. 6. An antibody or HTT-binding fragment of said antibody according to any one of claims 1 and 3-5, which specifically binds an epitope in the C-terminal region encoded by exon 1 of HTT, which contains the amino acid sequence PPGPAVAEEPLHRP (SEQ ID NO: 145). 7. An antibody or HTT-binding fragment of said antibody according to any one of claims 2 to 5, which specifically binds an epitope in the C-terminal region encoded by exon 1 of HTT, which contains the amino acid sequence PPGPAVAEEPLH (SEQ ID NO: 202). 8. An antibody or an HTT-binding fragment of said antibody according to any one of claims 1 to 7, characterized in that said constant domain is a human immunoglobulin constant domain of the IgG type. 9. An antibody or HTT-binding fragment of said antibody according to claim 8, wherein said human immunoglobulin constant domain is a constant domain of the IgG1 class or isotype. 10. An antibody or HTT-binding fragment of said antibody according to any one of claims 1 to 9, characterized in that said antibody more preferentially recognizes mutated, aggregated and/or soluble forms of HTT than physiological forms of HTT. 11. An antibody or HTT-binding fragment of said antibody according to any one of claims 1 to 10, which is a chimeric mouse-human antibody or an antibody containing elements of mouse antibodies, and/or an antibody or HTT-binding molecule that competes with the antibody according to any from claims 1-5 for specific binding to HTT. 12. A polynucleotide encoding an antibody or an HTT-binding fragment of said antibody according to any one of claims 1, 3-6 and 8-11 or its VH and VL chains. 13. A polynucleotide encoding an antibody or an HTT-binding fragment of said antibody according to any one of claims 2-5 and 7-11 or its VH and VL chains. 14. A vector for expressing an antibody or an HTT-binding fragment of said antibody according to any one of claims 1 to 11, or the VH and VL chains thereof, wherein said vector contains a polynucleotide according to claim 12 or 13. - 89 046697 15. A host cell for expressing an antibody or an HTT-binding fragment of said antibody according to any one of claims 1 to 11 or the VH and VL chains thereof, containing a polynucleotide according to claim 12 or 13 or a vector according to clause 14. 16. A method for producing an antibody or an HTT-binding fragment of said antibody according to any one of claims 1 to 11 or its VH and VL chains, wherein said method includes: (a) culturing the cell according to claim 15; and (b) isolating said antibody, the HTT-binding fragment or the VH and VL chains thereof from said culture. 17. An antibody or an HTT-binding fragment of said antibody encoded by the polynucleotide of claim 12 or 13 or obtained by the method of claim 16. 18. A bispecific antibody comprising an antibody according to any one of claims 1-11 or 17, wherein said antibody recognizes two different epitopes on a protein encoded by exon 1 of the HTT gene, wherein at least one epitope is selected from the group consisting of epitopes that are defined in paragraph 6 or 7. 19. A conjugate of an antibody or an HTT-binding fragment of said antibody according to any one of claims 111 and 17 or a bispecific antibody according to claim 18 with a detectable label, wherein said detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore and heavy metal. 20. A conjugate of an antibody or an HTT-binding fragment of said antibody according to any one of claims 111 and 17 or a bispecific antibody according to claim 18 with a drug. 21. A composition for binding HTT, containing an antibody or an HTT-binding fragment of said antibody according to any one of claims 1 to 11 and 17, or a bispecific antibody according to claim 18, or a conjugate according to claim 19 or 20, and a carrier. 22. The composition according to claim 21, which is a pharmaceutical composition, and said carrier is a pharmaceutically acceptable carrier. 23. The composition according to claim 21, which is a diagnostic composition containing standard reagents for use in immunodiagnostic or nucleic acid-based diagnostic methods. 24. Use of an antibody or HTT-binding fragment of said antibody according to any one of claims 1-11 and 17, a bispecific antibody according to claim 18 or a conjugate according to claim 19 or 20 for visualizing HTT or targeting a therapeutic and/or diagnostic agent to HTT in in the human or animal body in vivo. 25. The use of claim 24, wherein said in vivo imaging comprises positron emission tomography (PET), single photon emission tomography (SPET), near-infrared (NIR) optical imaging, or magnetic resonance imaging (MRI) . 26. A kit for diagnosing a disorder associated with HTT amyloidosis, comprising at least one antibody or HTT-binding fragment of said antibody according to any one of claims 1 to 11 and 17, or a bispecific antibody according to claim 18, or a conjugate according to claim. 19 and additionally containing reagents and instructions for use.