Anti-human respiratory syncytial virus (RSV) antibodies and methods of use

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

Active Publication Date: 2013-05-08
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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  • Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
  • Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
  • Anti-human respiratory syncytial virus (RSV) antibodies and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0715] Expression of embodiment 1 RSV F protein

[0716] 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, recombinant RSV F protein was cloned and expressed in 293F cells. In a second example, the native RSV F protein was expressed by infecting HEp-2 cells with the RSV A2 strain.

[0717] A. Recombinant RSV F protein

[0718] In this example, the gene encoding the RSV F protein from the A2RSV strain was cloned and expressed. The RSV A2F gene (SEQ ID NO: 21 ) containing only the extracellular domain was synthesized according to standard DNA synthesis methods from GeneArt (Burlingame, CA). The RSV A2F gene was engineered to include a Kozak sequence (nucleotides 7-16 of SEQ ID NO:21), a c-myc sequence (nucleotides 1600-1629 of SEQ ID NO:21) and a 6X-His tag (SEQ ID NO : n...

Embodiment 2

[0724] Example 2 Isolation of anti-RSV Fab antibodies from EBV-transformed B cells

[0725] 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. Sections A and B describe two slightly different methods for generating and cloning EBV-transformed B cells and screening for binding to RSV F protein. The differences are as follows: (1) the source of PMBC in step 1; (2) the isolation of IgG+B cells in step 2 (IgM, IgD and IgA expressing cells were removed in section A, and CD3 positive cells were additionally removed in section B); and (3) preparation of B-cell-depleted feeder cells irradiated in step 3.a.

[0726] A. Generation and cloning of EBV-transformed B cells and screening for binding to RSV F protein (Method A)

[0727] Peripheral blood mononuclear cells (PMBC) were obtained from child care workers...

Embodiment 3

[0866] Embodiment 3 separates anti-RSV Fab antibody by single cell sorting

[0867] 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.

[0868] B cell isolation

[0869] 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 to magnetically label and remove 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. By using biotin-conjugated monoclonal antibody (biotin-antibody mixture) as primary labeling reagent and anti-bioti...

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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] U.S. Provisional Application No. 61 / 399,310, entitled "Antibodies against Human Respiratory Syncytial Virus (RSV) and Methods of Use," by Robert Anthony Williamson, Jehangir Wadia, Gabriel Pascual, Elissa Keogh, filed July 9, 2010, and Priority to U.S. Provisional Application No. 61 / 456,454, entitled "Anti-Human Respiratory Syncytial Virus (RSV) Antibodies and Methods of Use," filed November 5, 2010, by Robert Anthony Williamson, Jehangir Wadia, Gabriel Pascual, and Elissa Keogh . The subject matter of each application cited above is incorporated herein by reference in its entirety. [0003] This application is related to U.S. Application Serial No. 12 / 806,498, filed August 13, 2010, published as US-2011-0076268 A1 and entitled "Antibodies Against Human Respiratory Syncytial Virus (RSV) and Methods of Use," and to the August 2010 International PCT Application No. WO 2011 / 020079 and International Application No. PCT / US10 / 045549, filed on the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10A61P31/12
CPCC07K2317/33C07K16/1027C07K2317/21C07K2317/76C07K16/10C07K2317/55C07K16/468A61K2039/505C07K2317/92C07K2317/31C07K2317/565C07K2317/56A61K31/7056A61P11/00A61P31/12A61P31/14A61K39/395A61K39/42A61K2039/507C07K2317/14C07K2317/24C12N7/00C12N2760/18534C12N2760/18571G01N33/56983G01N2333/135A61K45/06A61K2300/00C07K2317/35
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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