Anti-tsg101 antibodies and their uses for treatment of viral infections

A-TSG101, a virus infection technology, applied in the detection of virus-infected cells and the treatment of virus infections including HIV infection, can solve the problem of drug resistance weakening the therapeutic potential of virus fusion inhibitors

Inactive Publication Date: 2010-02-03
ELI LILLY & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, despite their antiviral potency and novel mechanism of action, drug resistance may weaken similar Therapeutic Potential of Viral Fusion Inhibitors

Method used

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  • Anti-tsg101 antibodies and their uses for treatment of viral infections
  • Anti-tsg101 antibodies and their uses for treatment of viral infections
  • Anti-tsg101 antibodies and their uses for treatment of viral infections

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0227] Example 1: Preparation and use of anti-TSG101 polyclonal antibody

[0228] To determine the effect of anti-TSG101 antibodies on viral infection, a retroviral infection assay was developed. Murine leukemia virus (MLV)-derived vectors including the E. coli lacZ gene expressed from the long terminal repeat (LTR) promoter (pBMN-Z-INeo) were transfected with facultative murine leukemia from 293 cells (Phoenix A, ATCC) Retroviral packaging cell lines. Retroviruses produced from Phoenix A helper cells were harvested and used to infect mouse N2A cells (ATCC). Anti-TSG101 antibody was added to 293 helper cells 24 hours after MLV vector transfection. The effect of TSG101 antibody on virus production was determined by the efficiency of viral supernatants to infect target cells (N2A). Infection of N2A cells was subsequently determined by cell staining for 3-galactosidase activity (positive cells stained blue, as figure 2 shown by dark dots in ).

[0229] Typically, phoenix ...

Embodiment 2

[0231] Example 2: Localization of TSG101 on the cell surface during virus budding

[0232] This example shows that the domain of TSG101 is exposed on the cell surface during HIV shedding and anti-TSG101 antibodies inhibit HIV shedding and infection.

[0233] Localization of TSG101 during viral shedding

[0234] In order to prove that TSG101 is actively involved in the release of the virus at the plasma membrane, the expression vector of the GFP-TSG101 fusion protein was constructed and transfected into Phoenix cells that actively produce M-MuLV virus (retroviral helper cell line developed by Nolan et al. ). 24 hours after transfection, the GFP-TSG101 fusion protein pathway was observed under a confocal microscope (Ultraview, Perkin-Elmer). Figure 3A ~E shows images of the time course of GFP-TSG101 protein translocation from the cytoplasm to the cell surface and then "budding" out of virus-producing cells.

[0235] Cell surface localization of TSG101 during HIV buddin...

Embodiment 3

[0246] Example 3: Effect of Anti-TSG101 Monoclonal Antibody on HIV Infection

[0247] Production of anti-TSG101 monoclonal antibody (mab)

[0248] A peptide consisting of 19 amino acids at the C-terminus of TSG101 (qlralmqkarktaglsdly, SEQ ID NO: 3) was synthesized, paired with keyhole limpet hemocyanin (KLH) and used to immunize 5 mice. Serum samples from each mouse were tested against BSA-conjugated C-terminal TSG101 peptide by ELISA. Mice with the highest antibody titers were killed and spleens fused with myeloma cells to generate a pool of hybridomas. PE-8 was one of a pool of hybridomas containing two hybridomas producing a mab that recognized the immunizing peptide (SEQ ID NO: 3). Pool PE-8 is also referred to as antibody pool PE-8.

[0249] Anti-TSG101mab inhibits wild-type and drug-resistant HIV

[0250] Briefly, wild-type and drug-resistant HIV expression vectors were transfected into HEK293 cells. 24 hours after transfection, transfected cells were treated...

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Abstract

The present invention provides antibodies that bind to the C-terminal region of TSG101. The invention also provides methods of using the TSG101 antibodies for the treatment of viral infections, including HIV and Ebola virus infection.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60 / 858,922, filed November 15, 2006, the disclosure of which is incorporated by reference in its entirety. This application is related to US Patent Application No. 11 / 939,122, filed November 13, 2007, also incorporated herein by reference. USSN 11 / 939,122 is directed to the preparation and use, especially of antibodies, of a family of proteins involved in viral budding, referred to herein as escort proteins. TSG101 is a member of this family. It is of unique importance. technical field [0003] The present invention relates to antibodies that bind to TSG101 protein and inhibit or reduce viral production. The invention also relates to methods of using TSG101 antibodies to treat viral infections, including HIV infection. The present invention further relates to methods for detecting virus-infected cells using the TSG101 antibod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/00C07K16/00
CPCC07K2317/34C07K2317/565C07K16/28C07K2317/732A61K2039/505C07K2317/76C07K16/18C07K2317/56C07K2316/96A61P31/12A61P31/14A61P31/18
Inventor 李利民迈克尔·金科迈克尔·戈德布拉特罗克珊·杜安
Owner ELI LILLY & CO
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