Human monoclonal antibody that binds/neutralizes botulinum neurotoxin type b

A technology of human monoclonal antibody and monoclonal antibody, which is applied in the fields of antibodies, antibacterial drugs, nervous system diseases, etc., and can solve the problems of prolonging outpatient rehabilitation treatment and long recovery

Active Publication Date: 2018-09-28
OSAKA UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recovery can be lengthy and involve extended outpatient rehabilitation

Method used

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  • Human monoclonal antibody that binds/neutralizes botulinum neurotoxin type b
  • Human monoclonal antibody that binds/neutralizes botulinum neurotoxin type b
  • Human monoclonal antibody that binds/neutralizes botulinum neurotoxin type b

Examples

Experimental program
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Effect test

Embodiment 1

[0174] Type B Clostridium botulinum The strain Okra was cultured using the cellophane-tube procedure to obtain a culture supernatant. The precursor toxins (16S toxin and 12S toxin) were purified from the culture supernatant using (Arimitsu et al., Infect. Immun. 71:1599-1603, 2003). BoNT / B1 and NAP-16 were prepared from 16S toxin as described (Arimitsu et al., Infect. Immun. 71:1599-1603, 2003). BoNT / A1 (strain 62A), BoNT / B2 (strain 111) and BoNT / B6 (strain Osaka05) were provided by Dr. S. Kozaki. Quadrivalent botulinum toxoid vaccine was provided by Dr. M. Takahashi. 12S toxin was purified from the respective culture supernatants of Clostridium botulinum types A, B, E, F and detoxified with formalin. Toxin detoxification was detected by intraperitoneal (i.p.) in mice. Four types of toxoid preparations were mixed together and then mixed with aluminum (adjuvant) and thimerosal (preservative). The toxoid preparation contained 0.1 mg of each of the four types, 0.2 mg alumin...

Embodiment 2

[0182] Cell fusion, selection and cloning procedures as described in figure 2 proceed as depicted in . Peripheral blood samples were collected from volunteers 9 or 18 days after the last immunization, and peripheral blood mononuclear cells (PBMC) were purified by Ficoll (GE Healthcare) gradient centrifugation. PBMC and SPYMEG cells were fused at a ratio of 10:1 with polyethylene glycol (Roche). Confluent cells were cultured in 96-well plates in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO) supplemented with 15% FBS in the presence of hypoxanthine-aminopterin-thymidine (HAT, GIBCO) About 10-14 days. After HAT selection, the medium was first screened for antibodies specific for BoNT / A or BoNT / B by ELISA. As a result, 27 positive wells were obtained from blood samples collected 9 days after inoculation, and 8 positive wells were obtained from blood samples collected 18 days after inoculation. Wells positive for specific antibodies are then subjected to cell cloning by lim...

Embodiment 3

[0194] Cloning and sequencing of variable region genes of monoclonal antibodies were performed. The sequences of the determined heavy and light chain variable regions are shown in Figure 3 ~ Figure 8 middle. Total RNA was extracted from hybridomas using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, using SUPERSCRIPT (R) VILO™ cDNA Synthesis Kit (Invitrogen) was generated by RT-PCR. The coding regions of the H- and L-chains of the M-2 and M-4 antibodies were amplified by PCR with KOD-Pus-Neo (Toyobo) and the following primers: 5′-ATGGACTGGACCTGGAGGATCCTC-3′(M-2-H- strand sense primer) (SEQ ID NO: 35), 5′-ATGAAACACCTGTGGTTTCTTCCTCCT-3′ (M-4-H-strand sense primer) (SEQ ID NO: 36), and 5′-CTCCCGCGGCTTTGTCTTGGCATTA-3′ (H-strand antisense primer) (SEQ ID NO: 38); and 5'-ATGGCCTGGWYYCCTCTCYTYCTS-3' (M-4-16-L-strand sense primer) (SEQ ID NO: 38), 5'-ATGSCCTGGGCTCYKCTSCTCCTS-3' (M -2- and M-4-18-L-chain sense primer) (SEQ ID NO: 39), 5'-ATGGCCTGGRYCYCM...

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Abstract

Isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragments thereof having a neutralization activity against a botulinum neurotoxin type B are provided, including a human monoclonal antibody. Hybridomas that produce such antibodies or fragments thereof are also provided by the present invention, as well as methods of producing such hybridomas and method of producing antibodies or fragments thereof from such hybridomas. Pharmaceutical compositions and kits including antibodies or fragments thereof for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning are further provided. Methods of inhibiting or treating botulism in a human subject are provided, as are methods of detecting botulinum neurotoxin type B.

Description

technical field [0001] The present invention relates to antibodies against botulinum neurotoxin (botulin) and methods of using and making them. [0002] [Related application] [0003] This application claims the benefit of US Provisional Patent Application No. 61 / 695318, filed August 31, 2012, which is hereby incorporated by reference in its entirety. Background technique [0004] Botulinum neurotoxin is the most poisonous substance known. These toxins can cause botulism and are caused by various bacteria including Clostridium botulinum produce. Clostridia, which produce botulinum toxin, are anaerobic, Gram-positive bacteria. These bacteria form spores and are often distributed in the environment. At the molecular level, botulism is caused by inhibition of acetylcholine release induced by the toxin at the neuromuscular junction. Seven toxin serotypes (A, B, C, D, E, F and G) have been identified. They have a similar structure to the zinc endopeptidase protein of appro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12A61K39/395A61P31/04C12N5/10G01N33/569C12P21/08
CPCA61K2039/505A61P25/00A61P31/04C07K16/1282C07K2317/21C07K2317/76G01N33/56911
Inventor 藤永由佳子松村拓大生田和良三崎亮藤山和仁幸田知子小崎俊司小野健一郎王荣萨·毗亚达
Owner OSAKA UNIV
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