Humanized antibody that recognizes α-synuclein
An antibody, human technology, applied in the direction of antibodies, immunoglobulins, anti-animal/human immunoglobulins, etc., can solve problems such as poisoning nerves
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Embodiment 1
[0180] Example 1. Design of Humanized 1H7 Antibody
[0181] The starting point of the humanized donor antibody is the murine antibody 1H7 produced by the hybridoma with ATCC deposit No. PTA-8220, which is described in US Patent Application No. 11 / 710248 and Publication No. US2009 / 0208487. The complete amino acid and nucleic acid sequences of the heavy chain variable region of m1H7 are shown in SEQ ID NO: 4 and 5, respectively. The complete light chain variable region amino acid and nucleic acid sequences of m1H7 are shown in SEQ ID NO: 6 and 7, respectively. The amino acid and nucleic acid sequences of the heavy chain variable region of mature m1H7 are shown in SEQ ID NO: 8 and 9, respectively. The amino acid and nucleic acid sequences of the light chain variable region of mature m1H7 are shown in SEQ ID NOS: 10 and 11, respectively. The amino acid sequences of heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 12, 13 and 14, respectively. The amino acid sequences of...
Embodiment 2
[0224] Example 2. Binding Kinetic Analysis of Murine, Chimeric and Humanized 1H7 Antibodies
[0225] The binding kinetics of humanized 1H7 antibodies comprising a heavy chain selected from Hu1H7VHv1-5 and a light chain selected from Hu1H7VLv1-4 were characterized.
[0226] Antibodies were analyzed using Biacore for full Biacore binding kinetics. For mouse 1H7 (such as Figure 3A shown), chimeric 1H7 (as shown Figure 3B shown) and humanized 1H7 (Hu1H7VHv3-Hu1H7VLv3, Hu1H7VHv3-Hu1H7VLv1, Hu1H7VHv4-Hu1H7VLv1) antibodies (such as Figure 3C The detailed binding kinetic parameters (association rate constant Ka, dissociation rate constant Kd and affinity constant KD) were determined. The binding kinetic parameters of humanized 1H7, especially Hu1H7VHv3-Hu1H7VLv3, were comparable to those of murine 1H7.
[0227] Table 3. Binding Kinetic Parameters of Murine 1H7, Chimeric 1H7 and Humanized 1H7
[0228] (Hu1H7VHv3-Hu1H7VLv3, Hu1H7VHv3-Hu1H7VLv1, Hu1H7VHv4-Hu1H7VLv1)
[0229] ...
Embodiment II
[0240] Example II. Passive immunization of α-synuclein antibodies
[0241]The purpose of this experiment was to determine the effectiveness of α-synuclein antibodies in in vitro and in vivo studies and behavioral assays. We used α-synuclein transgenic (row 61), α-synuclein knockout and wild-type female mice, initially 3-4 months old and n=14 / group. Antibodies detected include 9E4 (IgG1, epitope: amino acids 118-126 of α-synuclein), 5C1 (IgG1, epitope: amino acids 118-126 of α-synuclein , C-linker), 5D12, IgG2 (SN118-126), 1H7, IgG1 (SN91-99) and IgG1 control antibody 27-1. Mice received a dose of 10 mg / kg for a total of 21 injections over a period of 5 months. In addition, animals were injected with a lentiviral vector (LV) expressing human α-synuclein via unilateral introduction of human α-synuclein (wt) to the hippocampus.
[0242] Readout antibodies included those from Chemicon (epitope: full-length α-synuclein), Millipore (epitope: full-length α-synuclein) and Neotope, ...
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