SUBSTANCES AND METHODS FOR THE USE IN PREVENTION AND/OR TREATMENT IN HUNTINGTON'S DISEASE

Abstract

Disclosed is an apheresis device including a solid carrier capable of being contacted with the blood or plasma flow, characterised in that the solid carrier includes one or several HTT-binding molecule(s) capable of adsorbing HTT or fragments thereof in a specific manner from plasma or blood or other HTT containing body fluids such as CSF.

Claims

1: An apheresis device, comprising a solid carrier capable of being contacted with the blood or plasma flow, wherein the solid carrier includes a huntingtin (HTT)-binding molecule.

2: The apheresis device according to claim 1, wherein the HTT-binding molecule is an anti-HTT antibody.

3: The apheresis device according to claim 1, wherein the HTT-binding molecule is a monoclonal antibody or an HTT-binding fragment thereof.

4: The apheresis device according to claim 3, wherein the HTT-binding fragment is selected from the group consisting of a Fab fragment, a Fd fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, and ScFv fragment.

5: The apheresis device according to claim 2, wherein the antibody is a polyclonal antibody.

6: The apheresis device according to claim 2, wherein the antibody is specific for the PRR region of HTT, the C6 region of HTT, or both.

7: The apheresis device according to claim 1, wherein the antibody is a monoclonal antibody selected from the group consisting of a human monoclonal antibody, a humanized monoclonal antibody, a bispecific monoclonal antibody, and a chimeric monoclonal antibody.

8: The apheresis device according to claim 2, wherein the antibody is a monoclonal antibody, which comprises a heavy chain variable region CDR1 comprising GYSFTDFY (SEQ ID No. 54), a heavy chain variable region CDR2 comprising IDPKNGDT (SEQ ID No. 55), a heavy chain variable region CDR3 comprising ATYYGYTMDY (SEQ ID No. 56), a light chain variable region CDR1 comprising SSVTSSY (SEQ ID No. 57), a light chain variable region CDR2 comprising STS (SEQ ID No. 58) a light chain variable region comprising HQYRRPPRT (SEQ ID No. 59).

9: The apheresis device according to claim 8, wherein the antibody is the monoclonal antibody PRR13.

10: The apheresis device according to claim 2, wherein the antibody is a monoclonal antibody, which comprises a heavy chain variable region CDR1 comprising GYTFTEYT (SEQ ID No. 66), a heavy chain variable region CDR2 comprising INPNNGGT (SEQ ID No. 67), a heavy chain variable region CDR3 comprising ASLDGRDY (SEQ ID No. 68), a light chain variable region CDR1 comprising QSLLNSRTRKNY SEQ ID No. 69),a light chain variable region CDR2 comprising WAS (SEQ ID No. 70) and a light chain variable region comprising KQSYNLLT (SEQ ID No. 71).

11: The apheresis device according to claim 10, wherein the antibody is the monoclonal antibody C6-17.

12: The apheresis device according to claim 2, wherein the antibody is a monoclonal antibody, which comprises a heavy chain variable region CDR1 comprising GFTFNTYA (SEQ ID No. 72), a heavy chain variable region CDR2 comprising IRSKSNNYAT (SEQ ID No. 73), a heavy chain variable region CDR3 comprising VRHGEYGNPWFAY (SEQ ID No. 74), a light chain variable region CDR1 comprising QSLVHSNGNTY (SEQ ID No. 75),a light chain variable region CDR2 comprising KVS (SEQ ID No. 76) and a light chain variable region comprising SQSTHVPYT (SEQ ID No. 77).

13: The apheresis device according to claim 12, wherein the antibody is the monoclonal antibody M1D1.

14: The apheresis device according to claim 1, wherein the carrier is a sterile and pyrogen-free column.

15: The apheresis device according to claim 1, comprising at least two different HTT-binding molecules.

Description

[0108] The invention is further disclosed by the following examples and the figures, yet without being limited thereto.

[0109] FIG. 1 shows immune-titer analysis by ELISA of peptides derived from the PRR region and caspase region 586 of human Huntingtin (indicated as PRR and C6, respectively) in comparison to the less immunogenic N-terminal and poly-Q region of Huntingtin.

[0110] FIG. 2 shows mouse immunsera induced by peptides derived from the PRR and caspase region 586 of human Huntingtin. A recombinant 610 amino acids N-terminal Huntingtin fragment was captured from extracts of transiently transfected HEK293 cells in ELISA setup and incubated with immunesera thereby showing binding and specificity of induced antibodies.

[0111] FIG. 3 shows that individual immunsera raised against immunization peptide p6776 provide comparable anti-peptide ELISA titers against the immunization peptide (indicated as log EC50).

[0112] FIG. 4 shows that the same immunsera of FIG. 3 show differences in anti recombinant Huntingtin signals as measured by protein capture ELISA (OD; anti recHTT610) thereby demonstrating individual variation of the immune response.

[0113] FIG. 5 shows no changes of Huntingtin signals (EM48) in peptide vaccine (p6771, p6773) treated R6/1 mice when comparing to KLH-carrier or PBS treated F.6/l mice or KLH-carrier-treated wild type mice (numbers indicate Corrected Optical Density [COD] using Huntingtin-specific mAB EM48; error bars=standard deviations; n=10).

[0114] FIG. 6 shows that in contrast to the results from FIG. 5, the number of Synaptophysin-marked synapses (using mAB SY38) containing mutated human HTT (marked by EM48) is significantly reduced (p=0,001) in peptide vaccine-treated R6/1 mice (p6771, p6773) when compared to KLH treated R6/1 mice suggesting a beneficial effect of peptide treatment.

[0115] FIG. 7 shows that using neuron-specific marker NeuN, R6/1 mice display a significant neuroprotective effect in basal ganglia in the peptide vaccine-treated groups (p6771, p6773) when compared to control groups treated with KLH or PBS.

[0116] FIG. 8 shows that GFAP staining of basal ganglia shows non-significant reduction of astroglial activation in peptide vaccine treated R6/1 animals (p6771, p6773) when compared to KLH and PBS controls, respectively.

[0117] FIG. 9 shows plasma Huntingtin determination by FRET analysis in wt and YAC128 transgenic animals, respectively, at 12 months after single vaccine, combinatorial vaccine or carrier control (KLH) treatment.

[0118] FIG. 10 shows Rotarod test in treated and control YAC128 mice measuring latency to fall (indicated as mean value in seconds; n=25 animals per group) performed at 4, 6, 8, 10 and 12 months (indicated as mean M4mean M12) in transgenic YAC128 mice treated with various single and combinatorial peptide vaccines as indicated.

[0119] FIG. 11 shows the core epitope of 5 p6776 vaccine-induced immunsera determined by alanine substitution scanning by peptide ELISA using indicated peptides containing single amino acid alanine substitutions.

[0120] FIG. 12 shows that mAB M1D1 specifically recognizes recombinant Huntingtin fragment with free aspartic acid 586 at the C-terminus stronger than recHTT610.

[0121] FIG. 13 shows that supernatants from hybridoma derived from mice immunized with peptide p6773 provide strong recognition of the immunization peptide in 7 out of 9 pre-screened candidate clones when tested by peptide ELISA.

[0122] FIG. 14 shows that only 2 out of 9 mAB candidates shown in FIG. 13, namely PRR13 and PRR18, specifically recognize recombinant Huntingtin when tested by recHTT610 capture ELISA.

[0123] FIG. 15 shows the specificity analysis of 4 preselected anti Huntingtin mAB's derived from peptide p7543 immunized mice.

[0124] FIG. 16 shows screening of preselected mABs by determination of specificity against cleaved peptide p7564 by peptide ELISA

[0125] FIG. 17 shows that mAB MIDI recognizes Huntingtin peptides from the caspase 6 cleavage region of at least 7AA length containing free C-terminal Aspartic Acid.

[0126] FIG. 18 shows specific depletion of recombinant Huntingtin recHTT610 from human serum using mABs PRR13 and C6-17 in comparison to isotype control antibody IgG.

[0127] FIG. 19-21 show recognition capacity of the antibodies used according to the present invention.

[0128] FIG. 22-23 show examples for antibody humanization.

EXAMPLES

Example 1:Identification of Peptides Targeting the Huntingtin N-Terminus

Animal Immunizations

[0129] Anti-HTT antibody inducing peptides were coupled to KLH carrier using GMBS as amine-sulfhydryl crosslinker (Thermo/Pierce, CatNr. 22309) according to standard recommended procedures for peptide coupling via Cystein. The conjugated peptide was formulated with Aluminium Hydroxide Gel adjuvant (1 g/ml final concentration; Alhydrogel; Brenntag, CatNr. 21645-51-2) using 30 g coupled peptide in a volume of 200 l per injection. Immunizations were typically performed in female BALB/c mice (typically 5 mice per group, aged 10 weeks) using above formulations. Control groups were immunized with non-conjugated KLH and/or PBS and adjuvant alone. Animals were vaccinated 3-6 times in regular intervals of 2 weeks and plasma or serum was collected one day before each boost and at final bleeding.

Peptide ELISA.

[0130] Peptide-induced immune responses in mice were determined by ELISA using heparin as anticoagulant. ELISA plates (Nunc Maxisorb) were coated with maleimide activated BSA as carrier to which Cystein containing peptides were coupled via stable thioether bonds. For titrations, plasma dilutions were added and peptide-specific antibodies were quantified by biotinylated anti-mouse IgG (Southern Biotech, CatNr. 1034-08) as detection antibody combined with Streptavidin-POD (Roche, CatNr. 1089153) and subsequent color reaction using ABTS. EC50 values were determined using curve fitting with a 4-parameter logistic using GraphPad Prism (GraphPad Software).

Generation of Cell Extracts Containing N-Terminal Huntingtin Fragment recETT610.

[0131] A DNA covering the coding region of the N-terminal 610 aminoacids of human Huntingtin protein extended by two C-terminal V5 tags were synthesized and cloned via XbaI and BamHI restriction sites into eukaryotic expression vector pCDH-EF1-MCS IRES Puro (SBI; CatNr. CD532A1) yielding plasmid precHTT610. Cloning procedures were performed according to standard molecular biology procedures essentially as indicated by manufacturers including restriction digestions and ligation reactions (NEB Quick ligase kit; CatNr. M2200L), bacterial transformation followed by clone selection and analysis. DNA fragment preparations from agarose gels were performed using standard DNA purification kits (Quiagen; CatNr. 27106). HEK293 freestyle cells (Invitrogen; CatNr. R790-07) were grown in medium as indicated by the manufacturer and transiently transfected with precHTT610 (or empty vector as control) using MAXreagent (Invitrogen; CatNr.16447-100) and Optimem (Gibco; CatNr.31985). 24-48 h after transfection, HEK cell lysates were obtained by cell lysis with NP-40 extraction buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pHS), aliquoted and stored at 80 C. Protein concentrations were determined using Qubit (Invitrogen; CatNr.Q32866) according to the manufacturer's instructions.

Detection of Huntingtin by Protein capture ELISA

[0132] Binding of antibodies to N-terminal fragment HTT610 was determined by a standard protein capture ELISA procedure using Maxisorb ELISA plates (Thermo; CatNr. 439454), coated with 50 l of a 1:5000 rabbit anti V5 mAB (Sigma, CatNr. V8137), blocking with blocking buffer (PBS, 1% BSA, 0,1% Tween 20), capturing of recombinant Huntingtin from HEK cell extracts (100 ng/l total protein) followed by incubation with several dilutions of mouse anti HTT sera (1:100; 1:300 and 1:900) or with mAB2166 as reference (diluted 1:2000; Millipore, Cat Nr. MAB2166) for 1 hour at RT. ELISA incubations, washing and detection procedures were performed according to standard procedures.

Affinity Purification of Antibodies from Plasma

[0133] Iodoacetyl-activated magnetic beads (BcMag; Bioclone CatNr. FG-102) were conjugated with cysteine-containing peptides according to the manufacture's protocol. After plasma/mAB incubation for 2 h at RT, beads were washed with high salt buffer (PBS, 0,2%. Triton X-100 supplemented to a final NaCl concentration of 350 mM), bound antibodies were recovered by acid elution (4 elution steps with 100 mM Glycine; pH2,8). After neutralization with a final concentration of 75 mM HEPES pH8, antibodies were concentrated to a volume of 100 l using Spin-X UF500 tubes (Corning, CatNr. CLS431478), protein concentration was measured as described for protein extracts.

Results:

[0134] Immune sera from Huntingtin peptide-immunized mice show that peptides derived from the polyproline rich region (PRR) and caspase region 586 (C6) of the Huntingtin protein generally provide higher titers in peptide ELISA analysis (FIG. 1) than comparable peptides derived from the polyglutamine (polyQ) or N-terminal region (comprising the first 17 amino acids) of the protein, respectively. When analysed by protein ELISA (FIG. 2), PRR- and caspase region 586-derived immunsera, show differences in anti-Huntingtin protein signal intensity (FIG. 4) and protein specificity (FIG. 2) depending on the peptide sequences of the immunization peptides, allowing for the definition of specific and immunogenic peptide candidates. Peptide p7564 induces immunsera specifically recognizing Huntingtin sequences containing Aspartic Acid at the C-terminus (FIG. 3) thereby providing a means for addressing a disease-specific Huntingtin neo-epitope generated by caspase cleavage of Huntingtin at position 586. FIG. 1: Immune-titer analysis by ELISA reveals that peptides derived from the PRR region and caspase region 586 of human Huntingtin (indicated as PRR and C6, respectively), provide on average titers above 1:10000 in peptide immunized mice, as opposed to animals immunized with polyglutamine region- or N-terminus-derived peptides (indicated as polyQ and Nter, respectively), where the average titers are below 1:10000. Titers are expressed as mean EC50 from 5 individual sera; error bars show standard deviations.

[0135] FIG. 2: Mouse immunsera from peptide derived from the PRR and caspase region 586 of human Huntingtin were screened by protein capture ELISA against a recombinant 610 aminoacids N-terminal Huntingtin fragment captured from extracts of transiently transfected HEK cells. Anti-Huntingtin (recHTT610) and background signals (CTRL) differ between different peptides despite a homogenous anti-peptide signal distribution as seen in peptide ELISA (shown in FIG. 1). Bars represent signals from 5 pooled immunsera each, tested either against recombinant Huntingtin (OD[recHTT610 extract]; light grey bars) or control extracts (OD[mock transfected ctrl extract]; dark grey bars), respectively at serum dilutions of 1:100.

[0136] FIG. 3: 5 Individual immunsera raised against immunization peptide p6776 provide comparable anti-peptide ELISA titers against the immunization peptide (indicated as log EC50).

[0137] FIG. 4: In contrast to anti-peptide titers (shown in FIG. 3), the same immunsera show differences in anti recombinant Huntingtin signals as measured by protein capture ELISA (OD; anti recHTT610) thereby demonstrating individual variation of the immune response.

Example 2:Peptide Immunization of Transgenic R6/1 Mice Overexpressing the First Exon of Mutant Human Huntingtin Provides Beneficial Changes Reflected by Neuropathological Markers in Basal Ganglia

[0138] R6/1 mice expressing exon 1 of human mutant Huntingtin under a relatively strong promoter (see Bard et al. 2014 and citations therein) were subjected to vaccine injections at week 8, 10, 14 and 24 formulated as in Example 1. For monitoring titers, plasma was collected at 8, 16, 28 and 32 weeks.

Immunohistochemistry

[0139] Analysis by immunohistochemistry was essentially performed as described in Mandler et al. 2014 [PMID: 24525765] using antibodies EM48, SY38, GFAP and NeuN for marker protein detection basal ganlia (Millipore, CatNr. MAB5374, MAB5258, AB5804 and MAB377, respectively).

Results:

[0140] Immunohistochemical analysis of basal ganglia of peptide immunized 6 months old transgenic R6/1 mice, overexpressing the first exon of mutant human Huntingtin. The effect of peptide immunisation was compared by histopathological comparison of peptide p6771 and p6773-immunized with control groups (KLH, PBS). A clear neuroprotective and Huntingtin-reducing effect in synapses was observed upon immunisation with PRR-derived vaccines thereby demonstrating that peptide-induced anti HTT antibodies are capable of providing a beneficial effect in vivo with respect to the HTT phenotype.

[0141] FIG. 5: No changes of Huntingtin signals (EM48) in peptide vaccine (p67.sup.71, p6773) treated R6/1 mice when comparing to KLH-carrier or PBS treated R6/1 mice or KLH-carrier-treated wild type mice (numbers indicate Corrected Optical Density [COD] using Huntingtin-specific mAB EM48; error bars=standard deviations; n=1.0).

[0142] FIG. 6: In contrast, the number of Synaptophysin-marked synapses (using mAB SY38) containing mutated human HTT (marked by EM48) is significantly reduced (p=0,001) in peptide treated R6/1 mice (p6771, p6773) when compared to KLH treated R6/1 mice (Student's ttest; n=10 animals per treatment group). Numbers indicate the ratio (in %) of SY38-positive synapses co-localizing with EM48 signals (error bars=standard deviations; COD=Corrected Optical Density).

[0143] FIG. 7: Using neuron-specific marker NeuN, R6/1 mice display a significant neuroprotective effect in basal ganglia in the peptide treated groups (p6771, p6773) when compared to control groups treated with KLH or PBS (p=0,002 and p=0,01, resp. Student's ttest; n=10).Wt KLH=wild type controls; numbers indicate Corrected Optical Density (COD); error bars=standard deviations.

[0144] FIG. 8: GFAP staining of basal ganglia shows non-significant reduction of astroglial activation in peptide treated R6/1 animals (p6771, p6773) when compared to KLH and PBS controls, respectively (COD=Corrected Optical Density; wt. KLH=wild type controls; error bars=standard deviations).

Example 3: Combinatorial Vaccine Treatment Leads to Reduced Plasma Huntingtin Levels in YAC128 Transgenic Mice Combined with Motoric Improvement as Measured by Rotarod Test in 4-12 Months Old Animals Thereby Demonstrating that Combined Peptide-Induced Anti HTT Antibodies are Capable of Providing a Beneficial Effect In Vivo with Respect to the HTT Phenotype

YAC128 Mouse Immunizations

[0145] Five cohorts of full length mutant human Huntingtin expressing YAC128 mice (see Bard et al. 2014 and citations therein) and WT control littermates were assembled consisting of 150 total YAC128 and 25 total WT. WT mice were treated with KLH control. YAC128 mice were divided into 6 treatment groups including 5 experimental peptide treatments and a KLH control group. Mice received treatments by s.c. injection at 1, 2, 3, 6 and 9 months of age as in Example 1. For combination immunization, the total peptide amount of 30 g per dose was kept by combining two peptides at 15 g+15 g each per 200 l volume dose.

Determination of Plasma Huntingtin Levels in Vaccine Treated YAC128 Mice

[0146] Plasma Huntingtin levels were determined by FRET (Frster resonance energy transfer)-based detection assay yielding the ratio between the two detection antibodies as previously described by Weiss et al. 2009 [PMID: 19664996]. The correlation between plasma HTT reduction by anti-HTT antibodies and the associated phenotypic changes in YAC128 mice provides evidence for the usefulness of an antibody-based therapeutic strategy for plasma HTT reduction. It is demonstrated that reduction of plasma HTT by peptide induced antibodies is beneficial, therefore it can be expected that the corresponding derived monoclonal antibodies are beneficial for a therapeutic apheresis approach in order to specifically reduce plasma HTT such as demonstrated here.

Rotarod Test

[0147] Two-month-old YAC128 mice were trained over 3 consecutive days on the rotarod (Ugo Basille) at a fixed speed of 18 revolutions per minute (RPM). Mice received 3120 s training trials per day with a 1 h inter-trial interval (ITI). Mice that fell from the rod were immediately replaced for the duration of the trial. The latency to the first fall and number of falls for each training trial were recorded. The average of the 3 trials for each mouse was scored. For longitudinal rotarod testing at 2 month intervals from 2 to 12 months of age, an accelerating program from 5 RPM to 40 RPM over 300 s was used. Mice received 3 trials with a 1 h ITI and the latency to the first fall was recorded. The average of the 3 testing trials was scored.

Results:

[0148] FIG. 9: Plasma Huntingtin determination by FRET analysis in wt and YAC128 transgenic animals, respectively, at 12 months after single immunization, combinatorial immunization or carrier control (KLH) treatment. Peptides from the PRR and caspase region 586 regions were used (p6771 and p7564&p7543, respectively). Significant reduction of plasma Huntingtin can be achieved by combinatorial treatment using peptide combinations p7543+p7564 or p7543+p6771, when comparing plasma Huntingtin levels to carrier control treatment (KLH) (p<0,001 and p<0,01, respectively; Student's ttest; n=25 animals per treatment group). Numbers indicate relative units (FRET); error bars indicate standard deviations.

[0149] FIG. 10: Rotarod test in treated and control YAC128 mice measuring latency to fall (indicated as mean value in seconds; n=25 animals per group) performed at. 4, 6, 8, 10 and 12 months (indicated as mean M4-mean M12) in transgenic YAC128 mice treated with various single and combinatorial peptide immunizations as indicated. Notably, the combinatorial groups p7543+7564 and p7543+p6771 showed overall better performance in this test when compared to single peptide treated groups p7543, p6771 and 7564, respectively. Motoric improvement was significantly improved at M4-M10 in combination group p7543+7564 when compared to carrier control groups (p<0,03, 0,02, 0,01, resp.; Student's ttest, n=25). This finding parallels plasma Huntingtin reduction as described in FIG. 9 and shows that reduction of HTT in the blood stream of a Huntington patient is beneficial.

Example 4: Epitope Mapping of Monoclonal and Polyclonal Antibodies Obtained by Immunisation with Peptides p6773, p7564 and p7543

Determination of Core Epitopes

[0150] Peptide epitope mapping was performed using alanine substitution scanning by determination of titer values (OD[EC50]) by ELISA as explained in Example 1 or alternatively by applying peptide microarrays as described by Stadler et al. 2008. In brief, peptides containing single alanine-substitutions each position of the peptide were spotted on the arrays, and the loss of signal due to substitutions at single positions was determined by fluorescence labelled secondary antibodies in combination with a Odyssey Imaging System by LI-COR Biosciences. This allowed for an evaluation of the contribution of each individual amino acid of the peptide to the epitope. Using this method, the original immunization peptide to be mapped plus single alanine-substituted variants for each individual position or the peptide were spotted onto microarrays and hybridized by the respective monoclonal antibodies or immune sera to be tested. When the resulting signal from an alanine-substituted peptide was reduced to less than 70% of the signal from the original immunization peptide, the respective alanine-substituted amino acid position was defined as part of the core epitope. Resulting core epitope sequences are provided below from individual sera or mAB's.

Results:

[0151] Polyclonal, affinity purified antibodies and monoclonal antibodies were derived from individual mice immunized with PRR-region derived peptides (including p6771 and p6773) and caspase region 586-derived peptides (including p7543 and p6776). Epitopes were mapped using alanine scanning. In brief, epitopes of individual sera and monoclonal antibodies were determined by testing antibodies against peptides with single amino acid substitutions for each position using either peptide microarrays or conventional peptide ELISA (as exemplified in FIG. 11)

Peptide and epitope alignments for PRR region-derived peptides p6771 and p6773 as determined by alanine substitution scanning:

TABLE-US-00006 (SEQIDNo.1) LPQPPPQAQPLLPC...... immunizationpeptidep6771 (SEQIDNo.4) LPQPPPQAQPLLPQPQPC.. immunizationpeptidep6773 (SEQIDNo.78) ..........LLPQP..... epitopemappedformABPRR13 (SEQIDNo.79) ....PPQAQPL......... epitopemappedforpolyclonalp6773serum1 (SEQIDNo.80) ....PPQAQP.......... epitopemappedforpolyclonalp6773serum2 (SEQIDNo.81) ........QPLL........ epitopemappedforpolyclonalp6773serum3 (SEQIDNo.82) .....PQAQPLL........ epitopemappedforpolyclonalp6773serum4
Peptide and epitope alignment of p7543 vaccine induced polyclonal immunsera and mAB C6-17 as determined by alanine substitution scanning:

TABLE-US-00007 (SEQIDNo.3) GTDNQYLGLQIGC immunizationpeptidep7543 (SEQIDNo.83) QYLGLQIG epitopemappedformonoclonalABC6-17 (SEQIDNo.84) YLGLQIG epitopemappedforpolyclonalp7543serum1 (SEQIDNo.85) DNQYLGLQIG epitopemappedforpolyclonalp7543serum2 (SEQIDNo.86) DNQYLGL epitopemappedforpolyclonalp7543serum3 (SEQIDNo.87) YLGLQIG epitopemappedforpolyclonalp7543serum4

[0152] Peptide and epitope alignments for caspase region 586 derived peptides spanning aspartic acid 586:

[0153] FIG. 11: The core epitope of 5 p6776 induced immunsera was determined by alanine substitution scanning by peptide ELISA using indicated peptides containing single amino acid alanine substitutions. The 5 sera (represented by dark to bright bars) were hybridized to alanine substituted peptides as indicated (for peptide sequences see table 1). As a result, 2 out of 5 animals showed signal reduction upon alanine substituted peptides p7754, p7756, p7757 and p7758, respectively, thereby delineating a core epitope with the amino acid sequence IVLD (SEQ ID NO:101). Numbers indicate the ration of titer OD (log EC.sub.50) [Ala-substituted peptide: wt-peptide].

[0154] Epitope mapping of p7564 induced antisera and mAB MIDI is provided in Example 5, FIG. 17.

[0155] FIG. 12: mAB MIDI specifically recognizes recombinant Huntingtin fragment with free aspartic acid 586 at the C-terminus stronger than recHTT610. Protein ELISA (performed as in Example 1) using recombinant Huntingtin with 610 and 586 amino acids length (HTT610 and HTT586, respectively). Values indicate the ratio of the mAB MIDI signal (OD; protein capture ELISA as explained in Example 1) to the mAB 2166 control antibody signal. Values were normalized to reference mAB 2166 recognizing an internal epitope present in both fragments as protein loading control.

Example 5. Generation and Characterisation of Monoclonal Antibodies PRR13, C6-17 and MID1

Monoclonal Antibodies

[0156] For the production and isolation of monoclonal antibodies, the ClonaCell-HY Hybridoma Cloning Kit (STEMCELL technologies, CatNr. 28411) was used according to the instructions of the manufacturer. In brief, hybridoma fusions were performed with myeloma cell line SP2-0 under HAT selection and supernatants were initially screened by peptide ELISA using the immunization peptide, respectively, and an irrelevant control peptide for background determination. In the case of M1D1, ELISA against peptide p6776 containing free C-terminal aspartic acid was used in order to determine specificity to cleaved peptide with free C-terminal aspartic acid as indicated in Example 5. Candidate mABs were affinity purified as described and tested against recHTT610 by protein ELISA as indicated in Example 1. The number of screened fusion clones was typically 500 for each fusion, respectively. For VL and VH region sequencing, mRNA from fusion clones was extracted, reverse transcribed using Oligo(dT) primers and PCR amplified using variable domain primers to amplify both the VH and VL regions. VH and VL products were cloned using standard PCR cloning procedures (Invitrogen, CatNr. K4560-01), transformed into TOP10 cells and screened by PCR for positive transformants. Selected colonies were picked and analyzed by DNA sequencing on an ABI3130xl Genetic Analyzer.

Affinity Purification of Antibodies

[0157] mABs and polyclonal antibodies were isolated from hybridoma supernatant (SN) and plasma, respectively using BcMag Todoacetyl activated magnetic beads (Bioclone, FG-102) to which cysteine containing peptides were linked according to the manufacture's protocol. After plasma/SN incubation for 2 h at RT, beads were washed with high salt buffer (PBS, 0.2% Triton X-100, supplemented with NaCl to a final concentration of 350 mM) and the bound antibodies eluted 4 times with acidic elution buffer (Thermo, CatNr. 21004). After neutralization in HEPES pH8 (75 mM end concentration), eluted antibodies were concentrated and buffer was exchanged to PBS to a volume 100 l using Spin-X UF500 tubes (Corning, CLS431478). Antibody concentrations were determined with the Qubit system (Invitrogen, CatNr.Q32866) according to the manufacturer's protocol.

Results:

[0158] Antibody PRR13 was generated by hybridoma technique using peptide p6773 as immunogen. Peptide p6773 shows beneficial neuroprotective effects in active immunization of R6/1 transgenic animals as shown in Example 2 and overlaps with p6771. PRR13 was selected from 9 preselected candidate mABs recognizing a PRR-derived peptide as shown in FIG. 11. Out of the candidate mABs listed in FIG. 13, PRR13 was selected based on its favorable signal/noise ratio when hybridized to recombinant HTT610 as shown in FIG. 12.

[0159] FIG. 13: Supernatants from hybridoma derived from mice immunized with peptide p6773 provide strong recognition of the immunization peptide in 7 out of 9 pre-screened candidate clones when tested by peptide ELISA.

[0160] FIG. 14: In contrast to specific anti peptide signals (FIG. 11), only 2 out of 9 mAB candidates, namely PRR13 and PRR18, specifically recognize recombinant Huntingtin when tested by recHTT610 capture ELISA (as explained in Example 1). These two candidates provided an outstanding signal to noise ratio (i.e. >=4; calculated reHTT610-specific signal: HEK ctrl extracts), and PRR13 was selected for epitope characterization (see Example 4) and variable chain sequencing (see below). PRR13 was determined as IgG subtype mouse IgG2a.

TABLE-US-00008 >PRR13VHConsensusAminoAcidSequence (SEQIDNo.62): MGWSWVMLFLLSGTGGVLSEVQLQQSAPELVKPGASVKMSCKASGYSFTD FYMKWVKQSHGKGLEWIGDIDPKNGDTFYNQKFKGRATLTVDKSSSTAYM QLNSLTTEDSAVYYCATYYGYTMDYWGQGTSVTVSSAKTTAPSVYPLAPV CGDTTGSSVTLGCLVKGYF >PRR13VLConsensusAminoAcidSequence (SEQIDNo.63): MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASLGERVTMTCTASSS VTSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISS MEAEDAATYYCHQYRRPPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTS GGASVVCFLNNFYPR

[0161] Antibody C6-17 was generated by hybridoma technique using peptide p7543 as immunogen. Peptide p7543 showed beneficial therapeutic effects in YAC128 transgenic animals as demonstrated in Example 3. Although anti recHTT610 signals were comparable between 4 preselected mABs from this screen as shown in FIG. 13, the signal to noise ratios differed significantly amongst these candidates as shown in FIG. 14 showing a recHTT610 capture ELISA (performed as in Example 1). Based on its specificity and IgG subtype (determined as mouse IgG2a), C6-17 was selected for epitope characterization (see Example 4) and variable chain sequencing:

[0162] FIG. 15: Specificity analysis of 4 preselected anti Huntingtin mAB's derived from peptide p7543 immunized mice. Values for 4 mAB candidates represent signal to noise ratios of recombinant Huntingtin-specific OD-signal against control extract (determined by protein capture ELISA as explained in Example 1). mAB C6-17 provides the best signal to noise ratio.

TABLE-US-00009 >C6-17VHConsensusAminoAcidSequence (SEQIDNo.60): MGWSCIMLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKTSGYTFTE YTMHWVKQSHGKSLEWIGGINPNNGGTRYNQKFKGKATLTVDRSSSTAYM ELRSLTSEDSAVYYCASLDGRDYWGQGTTLTVSSAKTTAPSVFPLA >C6-17VLConsensusAminoAcidSequence (SEQIDNo.61): MVLMLLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTR KNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQ AEDLAVYSCKQSYNLLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGA SVVCFLNNFYPK

[0163] Antibody M1D1 was generated by hybridoma technique using peptide p7564 as immunogen. Peptide p7564 shows beneficial therapeutic effects in YAC128 transgenic animals as demonstrated in Example 3. Monoclonal antibody M1D1 was selected by differential screening of binding to peptides containing a free aspartic acid at the C-terminus against a peptide containing this Aspartic Acid residue embedded within the sequence such as e.g. p6776, as shown in Example 1, FIG. 3. Based on its specificity for the cleaved sequence, monoclonal antibody M1D1 was selected for further epitope characterization and variable chain sequencing. It was typed as mouse IgM and it binds to the neo-epitope of a human Huntingtin fragment that is generated upon cleavage of the protein or a corresponding peptide sequence by caspase 6 or any other protease cleaving at amino acid position D586.

[0164] FIG. 16: Screening of preselected mABs by determination of specificity against cleaved peptide p7564 by peptide ELISA (as explained in Example 1). In contrast to e.g. Ml-C2, mAB M1D1 shows the most favorable p7564 to p6776 OD signal ratio. M1D1 is therefore specific to the neo-epitope generated by proteolytic cleavage at position 586. The C-terminal aspartic acid of p7564 corresponds to the C-terminal cleavage point generated by caspase 6 and possibly other caspases. It thereby provides the means for specific cleavage detection at this site in analogy to polyclonal antisera generated with therapeutically beneficial peptide p7564 as shown in Example 3.

[0165] FIG. 17: mAB M1D1 recognizes Huntingtin peptides from the caspase 6 cleavage region of at least 7AA length containing free C-terminal Aspartic Acid. In contrast, shorter peptides or peptides without free C-terminal Aspartic Acid are not or only weakly recognized by MIDI thereby demonstrating specificity of this monoclonal antibody for the cleaved sequence with free COOH-terminal aspartic acid such as the free amino acid position 586 of cleaved human Huntingtin protein (Bars represent OD from peptide ELISA at a mAB concentration of 1 ng/l; peptide designations from left to right are as follows: p7564, p7562, p7552, p7541, p7567, p7568, p7605, p6777).

TABLE-US-00010 >M1D1VHConsensusAminoAcidSequence (SEQIDNo.64): MDFGLSWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFTFNT YAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSML YLQMNNLKTEDTAMYYCVRHGEYGNPWFAYWGQGTLVTVSAESQSFPNVF PL >M1D1VLConsensusAminoAcidSequence (SEQIDNo.65): MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVH SNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLT SGGASVVCFLNNFYPK

Example 6Use of Monoclonal Antibodies PRR13 and C6-17 for Depletion of Huntingtin Protein from Human Serum

Huntingtin Depletion

[0166] 100 g paramagnetic Streptavidin coated beads (Dynabeads T1, CatNr. 65601) were incubated in a buffer volume of 50 l with 20 ng/l biotinylated mABs for 1 h at RT. Human serum was diluted 1:4 with PBS and spiked with protein extracts from recHTT610 and mock transfected cells at a final extract concentration was 50-100 ng/l, respectively obtained as indicated in Example 1. Biotinylated mAB's were coupled to streptavidin beads and incubated o/n at RT with Huntingtin cell extract containing serum. After stringent bead washing (50 mM Tris, 250 mM NaCl, 0.1% Tween), to confirm recHTT610 depletion efficacy by monoclonal antibodies, input serum (containing recHTT610), mock serum and depleted recHTT610 containing serum was tested by recHTT610 capture ELISA as in Example 1.

Results:

[0167] In order to demonstrate that mAbs PPR13 and C6-17 can be utilized as specific adsorbers for therapeutic apheresis of plasma Huntingtin, excess recombinant Huntingtin recHTT610 was added to human sera from healthy donors and subsequently submitted to ex vivo depletion by biotin-immobilized mAB PRR13, C6-17 and control IgG, respectively. As shown in FIG. 18, monoclonal antibodies PRR13 and C6-17 but not IgG efficiently depleted excess Huntingtin from human plasma. These antibodies therefore provide convenient adsorbers for therapeutic apheresis for the treatment of Huntingtin. The usefulness of these antibodies for therapeutic apheresis treatment in Huntington's disease is supported by the fact that mAB PPR13 and C6-17 were generated by using p6773 and p7543 as immunization peptide. Peptides from these targeting regions are capable of inducing antibodies that reduce the Huntington's disease phenotype in transgenic animal models as shown in Example 2 and 3, respectively. In analogy corresponding mABs are expected to provide a similar benefit by depleting HTT as demonstrated in FIG. 18.

[0168] FIG. 18: Specific depletion of recombinant Huntingtin recHTT610 from human serum using mABs PRR13 and C6-17 in comparison to isotype control antibody IgG. As reflected by reduction of the Huntingtin signal in plasma (determined by protein ELISA such as in Example 1), these mAb's can efficiently adsorb and reduce Huntingtin as required for therapeutic apheresis. The graph represents the amount of Huntingtin signal (as OD; measured by protein capture ELISA) detected prior depletion (Input), after depletion by specific antibodies (mAB PRR13 and C6-17) and after depletion by an isotype control antibody (IgG).

Table 1: Preferred peptide uses (peptide name/peptide region/peptide sequence (C is for coupling to carrier protein; can be provided at N- or C-terminus of the peptide), except for p7564, p7541, p7552, p7562, p7563, p7567 or p7568, where a free C-terminal aspartic acid is required for the epitope); peptide list indicating name designations, mapping to protein region (region: Nter=N-terminus, polyQ=polyglutamine stretch, PRR=poly proline rich region, Ex1=mapping to exon 1, C6=caspase cleavage 586 region) and amino acid sequences (single letter code; Nter>Cter; a=beta-alanine; b=biotin)

Example 7Use of mAB PRR13 or mAB C6-17 as Adsorbers for Therapeutic Apheresis

[0169] Biotinylated anti HTT antibodies mAB PRR13, mAB C6-17 and an irrelevant control antibody (ctrl mAB) were immobilized on magnetic streptavidin beads and incubated with 1:1 PBS diluted human serum spiked with cell extract from transiently transfected HEK cells expressing recombinant HTT610 (indicated by HTT) or with control extract from non-transfected HEK cells (indicated ctrl). Adsorber beads coated by mAB PRR13 or mAB C6-17 showed recHTT capturing, respectively (FIG. 19; indicated PRR13 and C6-17; y-axis shows OD resulting from anti V5 detection of captured recHTT610 protein). Irrelevant CTRL extracts (ctrl) or beads coated by an irrelevant antibody (ctrl mAB) provided background controls. As a conclusion, this provides an example for the use of these antibodies as adsorber for HTT present in serum or other fluids as required for e.g. therapeutic apheresis.

[0170] Method: HTT Depletion

100 g paramagnetic Streptavidin beads (Dynabeads T1, CatNr. 65601) were incubated in a buffer volume of 50 l with 20 ng/l biotinylated mABs for 1 h at RT. Human serum was diluted 1:4 with PBS and spiked with protein extracts from recHTT610 and mock transfected HEK293 cells at a final extract concentration of 50-100 ng/l, prepared as in Example 1. Biotinylated mAB's were coupled to streptavidin beads and subsequently incubated with recHTT610-extract containing serum o/n at RT. To confirm recHTT610 depletion efficacy, input serum (containing recHTT610), mock serum and recHTT610-depleted serum was using recHTT610 capture ELISA as in example 1. Rec HTT610 depletion of serum by antibody-coated beads was quantified using an on-beads ELISA setting as follows: To detect bound recHTT610, beads were washed stringently using a 50 mM Tris, 250 mM NaCl, 0.1% Tween buffer followed by incubation with a rabbit V5 antibody (1:5000).

Example 8Use of Supplementary Example #1b: Monoclonal or Polyclonal Antibodies from the Present Invention Recognize Also Mutant HTT (mutHTT) Containing an Extended 82 Amino Acids Long Poly Q Stretch (mutHTT610 Q82)

[0171] A supernatant derived from HEK cells transiently expressing recombinant mutHTT610 Q82 was directly coated on ELISA plates and subsequently incubated with mAB PRR13 (5 ng/l), polyclonal p9395 serum mix (1:100 dilution; derived from 5 immunized animals) and an irrelevant mAB (5 ng/l). Results are depicted in FIG. 20. The Y-axis shows OD values obtained from ELISA using mAB PRR13, pooled serum from five p9395-immunized animals and irrelevant mAB, as indicated, whereby the same ELISA protocol as in Example 1 was applied. To determine the background signals of the ELISA assay, mAB PRR13 and pooled p9395 serum was incubated with 1.25 ng/ml cell extract as indicated.

Example 9: Derivatization of Recombinant Fab's Based on the Sequence Information of mAH PRR13 and C6-17, Respectively

[0172] PRR13 FAB and C6-17 FAB were transiently transfected in to HEK cells and tested by HTT capture ELISA using cellular extracts from HTT610 transfected HEK cells as shown above. mAB 2166 (1:2000) was used as positive control antibody. The results are depicted in FIG. 21. Binding signals (OD values on y-axis; light grey bars) for FAB PRR13 and FAB C6-17 are shown in direct comparison with anti HTT reference antibody mAB 2166 as indicated on the x-axis. Background signals were determined using control extracts (CTRL extr) derived from non-transfected HEK cells are indicated by dark grey bars. This exemplifies that a derivative of either mABs can also be used as adsorbers.

[0173] MethodFAB Expression:

[0174] Light chains and heavy chains (w/o the hinge region of IgG) of antibodies PRR13 and C6-17 were cloned into a CMV driven gene expression vector in order to obtain recombinant FAB in the supernatant (SN). SNs were collected after 24 to 48 h post transfection and the expression and binding functionality of these constructs was confirmed by peptide ELISA and rec HTT610 ELISA.

Example 10Antibody Humanization

[0175] a) Antibody humanization of original antibodies PRR13 and hC6-17, respectively was performed as follows: Prototypic frameworks for heavy and light chain variable regions were used for the generation of series hPRR13-1 to -16 and hC6-17-1 to -16, respectively. Series included prototypic variants containing modifications at one or several amino acid positions in the heavy (designated Framework H) and/or light chain (designated Framework L) as indicated in FIG. 22. Numbers reflect amino acid positions within the framework regions indicated for the humanized antibodies below.

TABLE-US-00011 hPRR13serieslightchainvariableregion (SEQIDNo.95) [EIVLTQSPSSLSASVGDRVTITCTASSSVTSSYLHWYQQKPGKAPKLLI YSTSNLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYRRPPRTF GGGTKLEIKR] hPRR13heavychainvariableregion (SEQIDNo.96) [EVQLVESGPEVKKPGATVKISCKVSGYTFTDFYMKWVQQAPGRGLEWMG DIDPKNGDTFYNQKFKGRVTMTADTSTGTAYMQLSSLTSEDTAVYFCASY YGYTMDYWGQGTTVTVAS]; hC6-17lightchainvariableregion (SEQIDNo.97) [DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYN LLTFGGGTKLEIK]; hC6-17heavychainvariableregion (SEQIDNo.98) [QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGRGLEWMG GINPNNGGTRYNQKFKGRVTMTRDTSIRTAYVELSRLTSDDTAVYYCASL DGRDYWGQGTLVTVSS]

[0176] Methods: Human vLC and vHC sequences were synthesized and cloned into the expression vector pFUSE2ss CLg-hk (EcoRI/NheI) and pFUSEss CHIg-hGl (EcoRI/BsiWI). Cloning procedures were performed according to standard molecular biology procedures essentially as indicated by manufacturers including restriction digestions and ligation reactions (NEB Quick ligase kit; CatNr. M2200L), bacterial transformation followed by clone selection and analysis. DNA fragment preparations from agarose gels were performed using standard DNA purification kits (Quiagen; CatNr. 27106). HEK293 freestyle cells (Invitrogen; CatNr. R790-07) were grown in medium as indicated by the manufacturer and transiently co-transfected with different combinations of hu AB heavy and light chain vectors as indicated in the table. Cell culture SNs were collected 24-48 h after transfection and concentrated 1:30 followed by buffer exchange (PBS) using Spin-X UF500 tubes (Corning, CLS431478). Concentrated human antibody-SNs were tested by in vitro peptide and protein binding using ELISA (as in Example 1). Further characterization was performed as indicated throughout this Example 9.

[0177] b) As an example, recognition of recHTT610 protein by humanized mAB PRR13 derivatives hPRR13-10, hPRR13-12 and hPRR13-14 (light grey bars) containing framework mutations as indicated in a) (see FIG. 22) can be demonstrated by protein ELISA (performed as in Example 1; see FIG. 23, y-axis reflects rec Htt610 binding activity[OD]) in comparison to a control extract (dark grey bars).

TABLE-US-00012 SEQID No. P6773 PRR LPQPPPQAQPLLPQPQPC 103 ++activevacc. mABgeneration p7564 C6 CPSDSSEIVLD 104 ++activevacc. mABgeneration p7543 C6 GTDNQYLGLQIGC 105 ++activevacc. mABgeneration C6inhibition p6771 PRR LPQPPPQAQPLLPC 106 ++activevacc. mABgeneration p8346 Ex1 CGPAVAEEPLHRP 107 ++activevacc. mABgeneration p8855 C6 SDSSEIVLDGTDC 108 ++activevacc. C6inhibition p8858 C6 EIVLDGTDNQYLC 109 ++activevacc. C6inhibition p8859 C6 IVLDGTDNQYLGC 110 ++activevacc. C6inhibition p8860 C6 VLDGTDNQYLGLC 111 ++activevacc. C6inhibition p8861 C6 LDGTDNQYLGLQC 112 ++activevacc. C6inhibition p8862 C6 DGTDNQYLGLQIGC 113 ++activevacc. C6inhibition p8869 C6 CTDNQYLGLQIGQ 114 ++activevacc. C6inhibition p8868 C6 CGTDNQYLGLQIG 115 +activevacc. C6inhibition p8870 C6 CDNQYLGLQIGQP 116 +activevacc. C6inhibition p8871 C6 CNQYLGLQIGQPQ 117 +activevacc. C6inhibition p6772 PRR CPQLPQPPPQAQPLLP 118 +activevacc. C6inhibition p8864 C6 TDNQYLGLQIGQC 119 ++activevacc. p8865 C6 DNQYLGLQIGQPC 120 ++activevacc. p6775 PRR PPPQLPQPPPQAQPLLPQPQPaC 121 ++activevacc. p8854 C6 PSDSSEIVLDGTC 122 +activevacc. p8856 C6 DSSEIVLDGTDNC 123 +activevacc. p8857 C6 SEIVLDGTDNQYC 124 +activevacc. p8866 C6 NQYLGLQIGQPQC 125 +activevacc. p8867 C6 QYLGLQIGQPQDC 126 +activevacc. p6763 Nter CaMATLEKLMKAFESLKSFQ 127 p6764 Nter CaKLMKAFESLKSFQ 128 p6765 polyQ CEEQQPQQQQQQQ 129 p6768 polyQ QQQQQQPPPPPPPPaKKKC 130 p7541 C6 CSEIVLD 131 p7552 C6 CSSEIVLD 132 p7562 C CDSSEIVLD 133 p7563 C6 CSDSSEIVLD 134 p7565 C6 CSEIVLDGT 135 p7567 C6 CEIVLD 136 p7568 C6 CIVLD 137 p7605 C6 CSEIVL 138 p6776 C6 CSEIVLDGTDNQYL 139 ++activevacc. C6inhibition p6777 C6 CSDSSEIVLDGTDN 140 ++activevacc. C6inhibition p6776b C6 SEIVLDGTDNQYLC 141 p7752 C6 CAEIVLDGTDNQYL 142 p7753 C6 CSAIVLDGTDNQYL 143 p7754 C6 CSEAVLDGTDNQYL 144 p7755 C6 CSEIALDGTDNQYL 145 p7756 C6 CSEIVADGTDNQYL 146 p7757 C6 CSEIVLAGTDNQYL 147 p7758 C6 CSEIVLDATDNQYL 148 p7745 C6 CSEIVLDGADNQYL 149 p7746 C6 CSEIVLDGTANQYL 150 p7747 C6 CSEIVLDGTDAQYL 151 p7748 C6 CSEIVLDGTDNAYL 152 p7749 C6 CSEIVLDGTDNQAL 153 p7750 C6 CSEIVLDGTDNQYA 154 Especially preferred for active vaccination (++active vacc.) Preferred for active vaccination (+active vacc.) Preferred for mAB generation (mAB generation) Preferred for C6 cleavage inhibition (C6 inhibition)

LITERATURE

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