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rsv-specific binding molecules

A specific, syncytial virus-based technology with applications in biology and medicine

Inactive Publication Date: 2020-07-28
MEDIMMUNE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, palivizumab is not always effective

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1. B cell culture, immortalization and selection.

[0135] method

[0136] As previously described (Scheeren FA, et al. (2005) Nat Immunol 6:303-313; Diehl SA, et al. (2008) J Immunol 180:4805-4815 and Kwakkenbos MJ, et al. (2009) Nat Med in press) Immortalization and culture of B cells. Briefly, we isolated B cells from peripheral blood by Ficoll separation, CD22MACS microbeads (Miltenyi Biotech), followed by CD19 sorting on a FACSAria (Becton Dickinson) + CD3 – CD27 + IgM – IgA – cells (IgG memory cells). Use of these tissues was approved by the Society Physician Ethics Committee and was based on informed consent. B cells transfected with retrovirus were supplemented with recombinant mouse IL-21 (25ng ml -1 , R&D system) in IMDM with 2x10 5 cell ml -1 Maintained, and with γ-irradiated (50Gy) murine L cell fibroblasts stably expressing CD40L (CD40L-L cells, 10 5 cell ml -1 ) co-cultured for 36 hours. BCL6 and Bcl-xL retroviral constructs were as pr...

Embodiment 2

[0140] Example 2. In vitro binding experiments.

[0141] In order to further confirm the antigen specificity of the AM22 antibody, we performed in vitro binding experiments to reveal whether the protein recognizes RSV F or G protein and what type of conformation it recognizes.

[0142] method

[0143] (1) FACS staining of RSV G or F protein

[0144] Prepare wild-type expression RSV-G protein (VSV-G) or RSV-F protein (VSV-F) on BHK cells cultured in DMEM containing 5% FCS, penicillin / streptomycin and 50 μM 2-mercapto-ethanol and virus stocks of recombinant vesicular stomatitis virus (VSV) (experimented by M. Lukens, WKZ, Utrecht, and VSV virus kindly provided by J.S. Kahn and J.K Rose, Yale University School of Medicine). VSV infection was performed on EL-4 cells cultured in Iscove's modified Dulbecco's medium (IMDM, Gibco, Invitrogen) supplemented with 5% FCS, penicillin / streptomycin and 50 μM 2-mercapto-ethanol. EL-4 cells infected with VSV virus variants were incubated ...

Embodiment 3

[0151] Example 3. In vivo potency experiments.

[0152] To study the in vivo potency of AM22 monoclonal antibody, cotton rat experiments were performed.

[0153] method

[0154] Pathogen-free 7–9 week-sized cotton rats (Sigmodon hispidus, Harlan Laboratories, Nederland) were anesthetized with isoflurane and administered 0.1 ml of purified antibody by intramuscular (i.m.) injection for the control antibody, palivizumab , AM22, AM23 and D25, 2.0 or 0.4mg kg -1 dose, while AM14 given 0.4 and 0.1mg kg -1 . Twenty-four hours later, the animals were anesthetized, blood was collected for serum hIgG determination and administered by intranasal instillation for 10 6 TCID 50 RSV-X (100 μl) challenge. The animals were sacrificed five days later to collect the lungs. Determination of lung virus titer and detection of the lowest limit of 2.1log 10 TCID 50 g-1. The Animal Testing Committee of the Netherlands Vaccine Society approved all procedures involving cotton rats.

[0155...

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Abstract

The invention provides an antibody capable of specifically binding RSV and functional equivalents of the antibody, and also provides a nucleotide sequence for encoding the antibody, and antibody producing cells and method for producing the antibody.

Description

[0001] This application is a divisional application of CN 200980162731.3 filed on 2009.10.06, titled "RSV-specific binding molecule". technical field [0002] The present invention relates to the fields of biology and medicine. [0003] Background of the invention [0004] Respiratory syncytial virus (RSV) is a cold virus belonging to the paramyxovirus family. RSV is virulent, easily contagious and is the most common cause of lower respiratory disease in children under 2 years of age. Up to 98% of day care children will be infected in one season of RSV infection. Between 0.5% and 3.2% of children with RSV infection require hospitalization. Reportedly, US hospitals receive about 90,000 cases and 4,500 deaths each year. The main risk factors for hospitalization due to RSV are premature birth, chronic lung disease, congenital heart disease, compromised immunity, and in otherwise healthy children younger than 6 weeks of age. No effective treatment is available for RSV-positiv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13
Inventor 蒂姆·博蒙特阿德里亚努斯·奎里纳斯·巴克安田悦子
Owner MEDIMMUNE LTD
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