Antibodies against human respiratory syncytial virus (RSV) and methods of use

A technology of antibodies and viruses, applied in the direction of respiratory diseases, antibodies, antiviral agents, etc., can solve problems such as vaccine treatment without RSV

Inactive Publication Date: 2012-09-05
JANSSEN VACCINES & PREVENTION BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no vaccine or commercially available treatment for RSV

Method used

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  • Antibodies against human respiratory syncytial virus (RSV) and methods of use
  • Antibodies against human respiratory syncytial virus (RSV) and methods of use
  • Antibodies against human respiratory syncytial virus (RSV) and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0641] Expression of embodiment 1 RSV F protein

[0642] In this example, the RSV fusion protein (F protein) from RSV strain A2 was expressed and captured on an ELISA plate using anti-RSV monoclonal antibody clone 2F7, which recognizes the F0 and F1 subunits of the fusion glycoprotein. Purify. In a first example, a recombinant RSV F protein comprising only the extracellular domain (SEQ ID NO: 25) was cloned and expressed in 293F cells. In a second example native RSV F protein (SEQ ID NO: 1629) was expressed by infecting HEp-2 cells with the RSV A2 strain.

[0643] A. Recombinant RSV F protein

[0644] In this example, the gene encoding the RSV F protein from the A2RSV strain was cloned and expressed. The RSVA2F gene (SEQ ID NO: 21 ) containing only the extracellular domain (full-length RSVA2F protein is shown in SEQ ID NO: 1630) was synthesized according to standard DNA synthesis methods at GeneArt (Burlingame, CA). The RSV A2F gene was engineered to include a Kozak sequen...

Embodiment 2

[0650] Example 2 Isolation of anti-RSV Fab antibodies from EBV-transformed B cells

[0651] In this example, anti-RSV antibodies were isolated from stimulated Epstein-Barr virus-transformed donor memory B cells, the cells were screened for binding to the RSV F protein, and then subjected to in vitro antibody production.

[0652] A. Purification of Donor Peripheral Blood Mononuclear Cells

[0653] Peripheral blood mononuclear cells (PMBC) were obtained from child care workers who were exposed to RSV through contact with children. PBMCs were isolated by density centrifugation on Ficoll Hypaque according to the manufacturer's instructions.

[0654] 1. CD22+ Isolation and Activation of CD22+ B Cells

[0655] 3.2x10 6 CD22+ B cells. In a 48-well plate, isolate CD22+ B cells at 1x10 6 cells / well were cultured in RPMI (Hyclone, cat.#SH30096.01), which contained 10% heat-inactivated low IgG fetal bovine serum (FBS, Invitrogen, cat.#16250-078), 1% antibiotics ( Hyclone, cat. # SV...

Embodiment 3

[0771] Embodiment 3 separates anti-RSV Fab antibody by single cell sorting

[0772] In this example, anti-RSV antibodies were isolated from CD19 / CD27 / IgG positive cells. CD19 / CD27 / IgG positive cells were obtained by 1) B cell isolation; and 2) FACS single cell sorting. The sorted cells are then used to isolate RNA that serves as a template for the production of Fab antibodies in vitro.

[0773] B cell isolation

[0774] B cells were isolated from PBMCs (harvested from anonymous blood bank donors) using a B cell isolation kit (Miltenyi Biotec, Cat. No. 130-091-151). The kit is used for magnetically labeling and removing cells expressing CD2, CD14, CD16, CD36, CD43 and CD235a (activated B cells, plasma cells and CD5+B-1a cells) and non-B cells (such as T cells, NK cells, dendritic cells, macrophages, granulocytes and erythroid cells) to isolate highly pure B cells. According to the manufacturer's protocol, by using biotin-conjugated monoclonal antibody (biotin-antibody mixtu...

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Abstract

Provided herein are antibodies or antigen-binding fragments thereof that immunospecifically bind to the fusion (F) protein of Respiratory Syncytial Virus (RSV). Also provided are methods for of prevention, treatment and diagnosis of viral infection and / or the treatment of one more symptoms of RSV-mediated disease. Methods of generating antibodies that immunospecifically bind RSV F protein also are provided.

Description

[0001] related application [0002] Priority is claimed to US Provisional Application Serial No. 61 / 274,395, entitled "Antibodies against Human Respiratory Syncytial Virus (RSV) and Methods of Use," filed August 13, 2009. [0003] This application is related to corresponding US Application Serial No. 12 / 806,498, entitled "Antibodies against Human Respiratory Syncytial Virus (RSV) and Methods of Use," which also claims priority to US Provisional Application Serial No. 61 / 274,395. [0004] The subject matter of each application cited above is incorporated herein by reference in its entirety. [0005] Incorporation by reference into electronically provided sequence listings [0006] An electronic version of the Sequence Listing is submitted with this application, the contents of which are incorporated herein by reference in their entirety. The electronic file is 602 kilobytes in size and titled 1152seq.PC1.txt. technical field [0007] Provided are antibodies that immunospecif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10A61K39/42A61P31/14G01N33/53A61K38/00A61K31/00A61K47/48C12N5/10C12N15/13C12N15/63
CPCC07K2317/21C07K2317/55C07K2317/76C07K16/1027A61P11/00A61P31/12A61P31/14
Inventor R·A·威廉森J·瓦迪亚G·帕斯夸尔E·基奥
Owner JANSSEN VACCINES & PREVENTION BV
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