Human-derived anti-huntingtin (htt) antibodies and uses thereof

Abstract

Provided are novel human-derived anti-huntingtin (HTT) antibodies and biotechnological derivatives thereof, preferably capable of binding mutated and/or aggregated HTT species and or fragments thereof, as well as methods related thereto. The human-derived anti-HTT antibodies and biotechnological derivatives can be used in pharmaceutical and diagnostic compositions for HTT targeted immunotherapy of Huntington Disease and diagnosis thereof.

1 . A human-derived monoclonal anti-huntingtin (HTT) antibody or an HTT-binding fragment, synthetic or biotechnological derivative thereof, which comprises variable domains of both the light (V L ) and heavy (V H ) chain and a constant domain. 
     
     
         2 . The antibody of  claim 1 , which is of the IgG type. 
     
     
         3 . The antibody of  claim 1  or  2 , wherein the light chain is kappa (κ). 
     
     
         4 . The antibody of any one of  claims 1  to  3 , which recognizes an epitope in the amino acid sequence of exon 1 of the HTT gene, preferably an epitope in a polyP-region, the P-rich-region, the C-terminal region, the N-terminal-region, the Q/P-rich-region or a conformational epitope of HTT exon 1. 
     
     
         5 . The antibody of any one of  claims 1  to  4 , which is capable of binding a peptide comprising the epitope and/or aggregated forms of HTT exon 1. 
     
     
         6 . The antibody of any one of  claims 1  to  5  or an HTT-binding fragment, synthetic or biotechnological derivative thereof which specifically binds
 (i) an epitope in a polyP-region which comprises the amino acid sequence PPPPPPPPPPP (SEQ ID Nos.: 146, 147, 148, 149, 150, 152, 153, 155, 156), PPPPPPPP (SEQ ID Nos. 139, 151, 154, 158, 161), PPPPPP (SEQ ID Nos.: 157, 159, 160); 
 (ii) an epitope in the P-rich-region which comprises the amino acid sequence PQPPPQAQPL (SEQ ID No. 140), PPPQLPQPPP (SEQ ID No. 141), QAQPLLPQPQPPPPP (SEQ ID No. 142), or PPPQLPQPPPQAQPL (SEQ ID No. 143); 
 (iii) an epitope in the C-terminal region of exon 1 of HTT which comprises the amino acid sequence PPGPAVAEEPLHRP (SEQ ID No.: 145); or PPGPAVAEEPLH (SEQ ID No. 202) 
 (iv) an epitope in the N-terminal region of exon 1 of HTT which comprises the amino acid sequence KAFESLKSFQQ (SEQ ID NO: 144); 
 (v) an epitope in the Q/P-rich-region which comprises the amino acid sequence QQQQQQQQQPPP (SEQ ID No. 201); or 
 (vi) a conformational epitope which is not present on a linear peptide derived from HTT exon 1 but on aggregated recombinant HTT exon1 proteins HttExon1Q21 (HD21) and HttExon1Q49 (HD49). 
 
     
     
         7 . The antibody of  claim 6  or an HTT-binding fragment, synthetic or biotechnological derivative thereof, which is selected from the group consisting of
 (i) an antibody recognizing a polyP-region of HTT comprising in its variable region
 (a) at least one complementarity determining region (CDR) of the V H  and/or V L  variable region amino acid sequence of any one of antibodies NI-302.33C11, NI_302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9 shown in  FIG. 1A, 1G, 1H, 1I, 1J, 1H, 1L, 1M, 1N, 10, 1P, 1Q, 1R, 1S, 1T, 1U, 1V, 1W, 1AD, 1AJ, 1AK ,  1 AL,  1 AM,  1 AN or  1 AO and depicted in
 (i) V H  sequence (SEQ ID NOs: 1, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89); and 
 (ii) V L  sequence (SEQ ID NOs: 3, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91), respectively; 
 
 (b) an amino acid sequence of the V H  and/or V L  region of any one of antibodies NI-302.33C11, NI_302.74C11, NI-302.15F9, NI-302.39G12, NI-302.11A4, NI-302.22H9, NI-302.44D7, NI-302.37C12, NI-302.55D8, NI-302.7A8, NI-302.78H12, NI-302.71F6, NI-302.11H6, NI-302.3D8, NI-302.18A1, NI-302.8F1, NI-302.52C9, NI-302.46C9 as depicted in  FIG. 1A, 1G, 1H, 1I, 1J, 1H, 1L, 1M, 1N, 10, 1P, 1Q, 1R, 1S, 1T, 1U, 1V, 1W, 1AD, 1AJ, 1AK ,  1 AL,  1 AM,  1 AN or  1 AO; 
 (c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a); 
 (d) a heavy chain and/or a light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); and/or 
 (e) the antibody or antigen binding fragment thereof optionally further comprising a polypeptide sequence which is heterologous to the V H  and/or V L  region or the least one CDR, preferably wherein polypeptide sequence comprises a human constant domain, preferably of the IgG type, most preferably of the IgG1 class or isotype; 
 
 (ii) an antibody recognizing the P-rich-region of HTT comprising in its variable region
 (a) at least one complementarity determining region (CDR) of the V H  and/or V L  variable region amino acid sequence of any one of antibodies NI-302.63F3, NI-302.31F11, NI-302.2A2, NI-302.15D3 or NI-302.64E5 shown in  FIG. 1B, 1D, 1E, 1X, 1AE, 1AG, 1AH, 1AQ, 1AR or 1AS  and depicted in
 (i) V H  sequence (SEQ ID NOs: 5, 13, 17, 135, 164, 166); and 
 (ii) V L  sequence (SEQ ID NOs: 7, 15, 19, 137, 168), respectively; 
 
 (b) an amino acid sequence of the V H  and/or V L  region of any one of antibodies NI-302.63F3, NI-302.31F31, NI-302.2A2, NI-302.15D3 or NI-302.64E5 as depicted in  FIG. 1B, 1D, 1E, 1X, 1AE, 1AG, 1AH, 1AQ, 1AR or 1AS ; 
 (c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a); 
 (d) a heavy chain and/or a light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); and/or 
 (e) the antibody or antigen binding fragment thereof optionally further comprising a polypeptide sequence which is heterologous to the V H  and/or V L  region or the least one CDR, preferably wherein polypeptide sequence comprises a human constant domain, preferably of the IgG type, most preferably of the IgG1 class or isotype; 
 
 (iii) an antibody recognizing the C-terminal-region of HTT exon 1 comprising in its variable region
 (a) at least one complementarity determining region (CDR) of the V H  and/or V L  variable region amino acid sequence of antibody NI-302.35C1 or NI-302.72F10 shown in  FIG. 1C, 1Z, 1AF or 1AT  and depicted in
 (i) V H  sequence (SEQ ID NO: 9, 176 or 178); and 
 (ii) V L  sequence (SEQ ID NO: 11, 180 or 182), respectively; 
 
 (b) an amino acid sequence of the V H  and/or V L  region of antibody NI-302.35C1 or NI-302.72F10 as depicted in  FIG. 1C, 1Z, 1AF or 1AT ; 
 (c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a); 
 (d) a heavy chain and/or a light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); and/or 
 (e) the antibody or antigen binding fragment thereof optionally further comprising a polypeptide sequence which is heterologous to the V H  and/or V L  region or the least one CDR, preferably wherein polypeptide sequence comprises a human constant domain, preferably of the IgG type, most preferably of the IgG1 class or isotype; 
 
 (iv) an antibody recognizing the N-terminal-region of HTT comprising in its variable region
 (a) at least one complementarity determining region (CDR) of the V H  and/or V L  variable region amino acid sequence of antibody NI-302.15E8 shown in  FIG. 1AP  and depicted in
 (i) V H  sequence (SEQ ID NOs: 131); and 
 (ii) V L  sequence (SEQ ID NOs: 133), respectively; 
 
 (b) an amino acid sequence of the V H  and/or V L  region of antibody NI-302.15E8 as depicted in  FIG. 1AP ; 
 (c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a); 
 (d) a heavy chain and/or a light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); and/or 
 (e) the antibody or antigen binding fragment thereof optionally further comprising a polypeptide sequence which is heterologous to the V H  and/or V L  region or the least one CDR, preferably wherein polypeptide sequence comprises a human constant domain, preferably of the IgG type, most preferably of the IgG1 class or isotype; 
 
 (v) an antibody recognizing the Q/P-rich-region of HTT comprising in its variable region
 (a) at least one complementarity determining region (CDR) of the V H  and/or V L  variable region amino acid sequence of antibody NI-302.7D8 in  FIG. 1Y  and depicted in
 (i) V H  sequence (SEQ ID NOs: 172); and 
 (ii) V L  sequence (SEQ ID NOs: 174), respectively; 
 
 (b) an amino acid sequence of the V H  and/or V L  region of antibody NI-302.7D8 as depicted in  FIG. 1Y ; 
 (c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a); 
 (d) a heavy chain and/or a light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); and/or 
 (e) the antibody or antigen binding fragment thereof optionally further comprising a polypeptide sequence which is heterologous to the V H  and/or V L  region or the least one CDR, preferably wherein polypeptide sequence comprises a human constant domain, preferably of the IgG type, most preferably of the IgG1 class or isotype; and 
 
 (vi) an antibody recognizing a conformational epitope which is not present on a linear peptide derived from HTT exon 1 but on aggregated recombinant HTT exon1 proteins HttExon1Q21 (HD21) and HttExon1Q49 (HD49) comprising in its variable region
 (a) at least one complementarity determining region (CDR) of the V H  and/or V L  variable region amino acid sequence of any one of antibodies NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1 shown in  FIG. 1F, 1AA, 1AB, 1AC or 1AU  and depicted in
 (i) V H  sequence (SEQ ID NOs: 21, 184, 188, 190, 194 or 196); and 
 (ii) V L  sequence (SEQ ID NOs: 23, 186, 192 or 198), respectively; 
 
 (b) an amino acid sequence of the V H  and/or V L  region of any one of antibodies NI-302.6N9, NI-302.4A6, NI-302.12H2 or NI-302.8M1 as depicted in  FIG. 1F, 1AA, 1AB, 1AC or 1AU ; 
 (c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a); 
 (d) a heavy chain and/or a light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); and/or 
 (e) the antibody or antigen binding fragment thereof optionally further comprising a polypeptide sequence which is heterologous to the V H  and/or V L  region or the least one CDR, preferably wherein polypeptide sequence comprises a human constant domain, preferably of the IgG type, most preferably of the IgG1 class or isotype. 
 
 
     
     
         8 . The antibody of any one of  claims 1  to  7 , wherein the antibody has a binding affinity corresponding to an EC50 (half maximal effective concentration) value of ≤20 nM, preferably ≤10 nM and most preferably ≤1 nM for binding HD49 and an EC50 value of 40 nM, preferably ≤10 nM and most preferably ≤1 nM for binding HD21. 
     
     
         9 . The antibody of any one of  claims 1  to  8  which is a chimeric murine-human or a murinized antibody and/or an antibody or HTT-binding molecule which competes with an antibody of any one of  claims 1  to  4  for specific binding to HTT. 
     
     
         10 . A polynucleotide encoding at least the binding domain or variable region of an immunoglobulin chain of the antibody of any one of  claims 1  to  9 , preferably wherein the polynucleotide is a cDNA. 
     
     
         11 . A vector comprising the polynucleotide of  claim 10 , optionally in combination with a polynucleotide of  claim 6  that encodes the variable region of the other immunoglobulin chain of said binding molecule. 
     
     
         12 . A host cell comprising a polynucleotide of  claim 10  or a vector of  claim 11 . 
     
     
         13 . A method for preparing an anti-HTT antibody, a biotechnological derivative, or immunoglobulin chain(s) thereof, said method comprising
 (a) culturing the cell of  claim 12  and   (b) isolating the antibody or immunoglobulin chain(s) thereof from the culture.   
     
     
         14 . An antibody or immunoglobulin chain(s) thereof encoded by a polynucleotide of  claim 10 , a vector of  claim 1  or obtainable by the method of  claim 13 . 
     
     
         15 . The antibody of any one of  claims 1  to  9  or  14 , which is a bispecific antibody. 
     
     
         16 . The antibody of  claim 15 , which recognizes two different epitopes on a protein encoded by exon 1 of the HTT gene, preferably wherein at least one epitope is selected form the group consisting of the epitopes as defined in any one of  claims 4  to  7 . 
     
     
         17 . The antibody of any one of  claims 1  to  9  or  14  to  16 , which is
 (i) detectably labeled, preferably wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, and a heavy metal; or 
 (ii) attached to a drug. 
 
     
     
         18 . A composition comprising the antibody of any one of  claims 1  to  9  or  14  to  17 , the polynucleotide of  claim 10 , the vector of  claim 11 , the cell of  claim 12 , preferably wherein the composition is
 (a) a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier, optionally further comprising an additional agent useful for treating diseases and/or symptoms associated with HTT amyloidosis, preferably wherein the composition is a vaccine; 
 (b) a diagnostic composition, preferably which comprises reagents conventionally used in immuno or nucleic acid based diagnostic methods. 
 
     
     
         19 . A HTT binding molecule comprising at least one CDR of an antibody of any one of  claims 1  to  9  or  14  to  17  for use in in vivo detection of or targeting a therapeutic and/or diagnostic agent to HTT in the human or animal body, preferably wherein said in vivo imaging comprises positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR), optical imaging or magnetic resonance imaging (MRI). 
     
     
         20 . A peptide having an epitope of HTT specifically recognized by an antibody of any one of  claims 1  to  9  or  14  to  17 , preferably wherein the peptide comprises an amino acid sequence as defined in  claim 2  or a modified sequence thereof in which one or more amino acids are substituted, deleted and/or added, wherein the peptide is recognized by the antibody of any one of  claims 1  to  9  or  14  to  17 . 
     
     
         21 . A kit useful in the diagnosis or monitoring of disorders associated with HTT amyloidosis, said kit comprising at least one antibody of any one of  claims 1  to  9  or  14  to  17  or a HTT binding molecule having substantially the same binding specificities of any one thereof, the polynucleotide of  claim 10 , the vector of  claim 11  or the cell of  claim 12  and/or the peptide of claim  220 , optionally with reagents and/or instructions for use. 
     
     
         22 . A method of treating a disease and/or symptoms associated with HTT amyloidosis or for in in vivo detection of or targeting a therapeutic and/or diagnostic agent to HTT in the human or animal body comprising administering to a subject in need thereof a therapeutically or diagnostically effective amount of an anti-HTT antibody or an HTT-binding fragment, synthetic or biotechnological derivative thereof, which comprises a variable light (V L ) and heavy (V H ) chain, and a constant domain for use in treating a disease and/or symptoms associated with HTT amyloidosis or for in in vivo detection of or targeting a therapeutic and/or diagnostic agent to HTT in the human or animal body. 
     
     
         23 . The method of  claim 22 , wherein the anti-HTT antibody is an antibody of any one  claims 1  to  9  or  14  to  17 .