Blood-Brain Barrier Shuttles Containing Antibodies Recognizing Alpha-Synuclein

Abstract

The invention provides blood-brain barrier shuttles comprising monoclonal antibody 5C1 and related antibodies linked to a monovalent binding entity for a blood-brain barrier receptor. The 5C1 antibody binds to an epitope within residues 118-126 of alpha-synuclein. The antibodies are useful, for example, for treating and/or diagnosing synucleinopathies including Lewy body diseases, such as Parkinson's disease, diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), Alzheimer's and Parkinson's disease comorbidity, and pure autonomic failure, as well as multiple system atrophy (MSA).

1 - 105 . (canceled) 
     
     
         106 . A blood-brain barrier shuttle comprising:
 (a) a humanized brain effector entity antibody comprising three heavy chain CDRs having amino acid sequences of SEQ ID NOS: 10, 11, and 12, respectively, and three light chain CDRs having amino acid sequences of SEQ ID NOS: 25, 26, and 27, respectively; and   ( ) a monovalent binding entity that binds to a blood-brain barrier receptor, the monovalent binding entity comprising an antibody fragment that is a scFv, Fv, scFab, sFab, or VHH and the blood-brain barrier receptor that is a transferrin receptor, insulin receptor, insulin-like growth factor receptor, low density lipoprotein receptor-related protein 8, low density lipoprotein receptor-related protein 1, or heparin-binding epidermal growth factor-like growth factor receptor;   wherein the brain effector entity is coupled to the monovalent binding entity by a first peptide linker.   
     
     
         107 . The blood-brain barrier shuttle of  claim 106 , wherein the monovalent binding comprises a sFab that specifically binds to a transferrin receptor. 
     
     
         108 . The blood-brain barrier shuttle of  claim 107 , wherein the monovalent binding entity binds to human transferrin receptor. 
     
     
         109 . The blood-brain barrier shuttle of  claim 107 , wherein the monovalent binding entity binds to an epitope within SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. 
     
     
         110 . The blood-brain barrier shuttle of  claim 109 , wherein the monovalent binding entity binds to an epitope consisting essentially of residues 10-15 of SEQ ID NO: 43, an epitope consisting essentially of residues 7-12 of SEQ ID NO: 44 or an epitope consisting essentially of residues 1-5 of SEQ ID NO: 45. 
     
     
         111 . The blood-brain barrier shuttle of  claim 107 , wherein the sFab comprises SEQ ID NO: 60. 
     
     
         112 . The blood-brain barrier shuttle of  claim 107 , wherein the monovalent binding entity comprises 8D3-scFab comprising a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 64 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 63, wherein the heavy and light chains of the 8D3-scFab are coupled by a second linker. 
     
     
         113 . The blood-brain barrier shuttle of  claim 107 , wherein the monovalent binding entity comprises a constant light chain region having an amino acid sequence comprising SEQ ID NO: 52, a constant heavy chain region having an amino acid sequence comprising SEQ ID NO: 65, and a variable light chain region and variable heavy chain region that specifically bind to human transferrin receptor. 
     
     
         114 . The blood-brain barrier shuttle of claim  2 , wherein the monovalent binding entity comprises a constant light chain region having an amino acid sequence comprising SEQ ID NO: 52, a constant heavy chain region having an amino acid sequence comprising SEQ ID NO: 65, and a variable light chain region and variable heavy chain region that bind to an epitope within SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. 
     
     
         115 . The blood-brain barrier shuttle of  claim 107 , wherein the monovalent binding entity comprises a variable and constant light chain region having an amino acid sequence comprising SEQ ID NO: 61 and a variable and constant heavy chain region having an amino acid sequence comprising SEQ ID NO: 62, wherein the variable and constant light chain region is coupled to the variable and constant heavy chain region by a peptide linker. 
     
     
         116 . The blood-brain barrier shuttle of  claim 106 , wherein the first and second peptide linkers have a length of at least 20 amino acids or about 25 to 50 amino acids. 
     
     
         117 . The blood-brain barrier shuttle of  claim 106 , wherein the first and second peptide linkers have an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 73. 
     
     
         118 . The blood-brain barrier shuttle of  claim 106 , wherein the brain effector entity antibody is of the isotype human IgG1. 
     
     
         119 . The blood-brain barrier shuttle of  claim 106 , wherein the brain effector entity antibody has at least one mutation in the constant region. 
     
     
         120 . The blood-brain barrier shuttle of  claim 119 , wherein the mutation reduces complement fixation or activation by the constant region compared to a brain effector entity antibody lacking the at least one mutation. 
     
     
         121 . A blood-brain barrier shuttle comprising:
 (a) a brain effector entity comprising an antibody comprising a heterodimerized heavy chain with first and second dimerization modules having amino acid sequences comprising SEQ ID NO: 53 and SEQ ID NO: 54, respectively, SEQ ID NO: 53 and SEQ ID NO: 69, respectively, or SEQ ID NO: 55 and SEQ ID NO: 56, respectively, or SEQ ID NO: 55 and SEQ ID NO: 70, respectively, wherein amino acid 1-115 of each first and second dimerization module comprises a mature heavy chain variable region having an amino acid sequence at least 90% identical to H4 (SEQ ID NO: 17) and comprising three heavy chain CDRs having amino acid sequences of SEQ ID NOS: 10, 11, and 12, respectively, and a mature light chain variable region having an amino acid sequence at least 90% identical to L3 (SEQ ID NO: 31) and comprising three light chain CDRs having amino acid sequences of SEQ ID NOS: 25, 26, and 27, wherein the antibody specifically binds to human alpha-synuclein; and   (b) a monovalent binding entity that binds to a blood-brain barrier receptor, the monovalent binding entity comprising an antibody fragment that is a scFv, Fv, scFab, sFab, or VHH and the blood-brain barrier receptor that is a transferrin receptor, insulin receptor, insulin-like growth factor receptor, low density lipoprotein receptor-related protein 8, low density lipoprotein receptor-related protein 1, or heparin-binding epidermal growth factor-like growth factor receptor;   wherein the brain effector entity is coupled to the monovalent binding entity by a peptide linker.   
     
     
         122 . The blood-brain barrier shuttle of  claim 121 , wherein the monovalent binding entity comprises a sFab that specifically binds to a transferrin receptor. 
     
     
         123 . The blood-brain barrier shuttle of  claim 122 , wherein the monovalent binding entity binds to human transferrin receptor. 
     
     
         124 . The blood-brain barrier shuttle of  claim 121 , wherein the monovalent binding entity binds to an epitope within SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. 
     
     
         125 . The blood-brain barrier shuttle of  claim 124 , wherein the monovalent binding entity binds to an epitope consisting essentially of residues 10-15 of SEQ ID NO: 43, an epitope consisting essentially of residues 7-12 of SEQ ID NO: 44 or an epitope consisting essentially of residues 1-5 of SEQ ID NO: 45. 
     
     
         126 . The blood-brain barrier shuttle of  claim 122 , wherein the sFab comprises SEQ ID NO: 60. 
     
     
         127 . The blood-brain barrier shuttle of  claim 122 , wherein the monovalent binding entity comprises 8D3-scFab comprising a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 64 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 63, wherein the heavy and light chains of the 8D3-scFab are coupled by a second linker. 
     
     
         128 . The blood-brain barrier shuttle of  claim 122 , wherein the monovalent binding entity comprises a constant light chain region having an amino acid sequence comprising SEQ ID NO: 52, a constant heavy chain region having an amino acid sequence comprising SEQ ID NO: 65, and a variable light chain region and variable heavy chain region that specifically bind to human transferrin receptor. 
     
     
         129 . The blood-brain barrier shuttle of  claim 122 , wherein the monovalent binding entity comprises a constant light chain region having an amino acid sequence comprising SEQ ID NO: 52, a constant heavy chain region having an amino acid sequence comprising SEQ ID NO: 65, and a variable light chain region and variable heavy chain region that bind to an epitope within SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. 
     
     
         130 . The blood-brain barrier shuttle of  claim 122 , wherein the monovalent binding entity comprises a variable and constant light chain region having an amino acid sequence comprising SEQ ID NO: 61 and a variable and constant heavy chain region having an amino acid sequence comprising SEQ ID NO: 62, wherein the variable and constant light chain region is coupled to the variable and constant heavy chain region by a peptide linker. 
     
     
         131 . The blood-brain barrier shuttle of  claim 121 , wherein the first and second peptide linkers have a length of at least 20 amino acids or about 25 to 50 amino acids. 
     
     
         132 . The blood-brain barrier shuttle of  claim 121 , wherein the first and second peptide linkers have an amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 73. 
     
     
         133 . The blood-brain barrier shuttle of  claim 121 , wherein the brain effector entity antibody is of the isotype human IgG1. 
     
     
         134 . The blood-brain barrier shuttle of  claim 121 , wherein the brain effector entity antibody has at least one mutation in the constant region. 
     
     
         135 . The blood-brain barrier shuttle of  claim 134 , wherein the mutation reduces complement fixation or activation by the constant region compared to a brain effector entity antibody lacking the at least one mutation. 
     
     
         136 . The blood-brain barrier shuttle of  claim 121 , wherein the brain effector entity antibody comprises a mature heavy chain variable region having an amino acid sequence at least 95% identical to H4 (SEQ ID NO: 17) and a mature light chain variable region at least 95% identical to L3 (SEQ ID NO: 31). 
     
     
         137 . The blood-brain barrier shuttle of  claim 121 , wherein the brain effector entity antibody's mature heavy chain variable region has an amino acid sequence designated H4 (SEQ ID NO: 17) and the brain effector entity antibody's mature light chain variable region has an amino acid sequence designated L3 (SEQ ID NO: 31). 
     
     
         138 . The blood-brain barrier shuttle of  claim 121 , wherein the brain effector entity antibody's mature heavy chain variable region has an amino acid sequence designated H5 (SEQ ID NO: 18) and the brain effector entity antibody's mature light chain variable region has an amino acid sequence designated L3 (SEQ ID NO: 31).