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Oncolytic adenoviral vectors coding for monoclonal anti-CTLA-4 antibodies

An oncolytic adenovirus, human monoclonal antibody technology, used in life sciences and medicine to achieve safe cancer treatment tools and improve the effect of cancer therapy

Inactive Publication Date: 2013-07-24
ONCOS THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, deletions of other regions as well as additional mutations have conferred additional properties to viral vectors

Method used

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  • Oncolytic adenoviral vectors coding for monoclonal anti-CTLA-4 antibodies
  • Oncolytic adenoviral vectors coding for monoclonal anti-CTLA-4 antibodies
  • Oncolytic adenoviral vectors coding for monoclonal anti-CTLA-4 antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1. Construction of adenovirus

[0159] Generation of chimeric adenoviruses with cDNA sequences encoding IgG2 type anti-CTLA4 mAbs ( figure 1 ). The coding sequence of the anti-CTLA4 mAb was introduced into the 6.7K / gp19K deletion of adenovirus E3A to produce replication-competent adenoviruses Ad5 / 3-Δ24aCTLA4 (SEQ ID.NO:1), Ad5 / 3-hTERT-Δ24aCTLA4 (SEQ ID.NO:2), Ad5 / 3-hTERT-Δ24aCTLA4-CpG (SEQ ID.NO:3), Ad5 / 3-E2F-Δ24aCTLA4 (SEQ ID.NO:4) and Ad5 / 3-E2F-Δ24aCTLA4-CpG (SEQ ID. NO: 5), or introduce a deletion at E1 driven by a CMV promoter to generate a replication-defective adenovirus Ad5 / 3-aCTLA4 (SEQ ID. NO: 6).

[0160] Using standard adenovirus preparation techniques (Kanerva A, et al., Mol Ther 2002; 5:695-704; Bauerschmitz GJ et al., Mol Ther 2006; 14:164-74; Kanerva A and Hemminki A., Int J Cancer 2004; 110:475-74; 80; Volk AL, et al., Cancer Biol Ther 2003;2:511-5) Generation and amplification of oncolytic adenovirus. Briefly, E1 or E3 shuttle vectors with t...

Embodiment 2

[0165] Example 2. In vitro expression and functionality of the constructed adenovirus

[0166] Western blot analysis was used to confirm that the constructed adenovirus expressed anti-CTLA4 mAb. A549 or PC3-MM2 tumor cells were infected with the constructed Ad5 / 3-Δ24aCTLA4 or Ad5 / 3-aCTLA4 at 10 viral particles (VP) / cell. After 48 hours, the supernatant of virus-infected cells was filtered with a 0.02 μm filter (Anotop, Whatman, England), and 15 μL was used for 7.5% SDS-polyacrylamide gel electrophoresis (PAGE ), and then transferred to nitrocellulose membrane. Membranes were incubated with goat anti-human IgG (heavy and light chains) (AbD serotec, MorphoSys, Germany), washed, and subsequently incubated with a secondary antibody conjugated to horseradish peroxidase (Dako, Denmark) . Signal detection was performed using enhanced chemiluminescence (GE Healthcare, Amersham, UK).

[0167] In Western blot, Ad5 / 3-Δ24aCTLA4 and Ad5 / 3-aCTLA4 expressed the expected anti-CTLA4 mAb of...

Embodiment 3

[0174] Example 3. CTLA-4 Expression in Tumor Cell Lines and Low-Passage Tumor Explants

[0175] Since it has been reported that almost 90% of tumor cell lines express CTLA-4 and that anti-CTLA-4 mAbs may have direct antitumor activity (13), it is important to study the expression of CTLA-4 in tumor cell lines, including HNSCC using low-passage tumor explants. Is this also the case in the body.

[0176] Indirect immunofluorescence was performed in the low passage tumor cell culture UT-SCC8 or the tumor cell lines A549, SKOV3-ip1 and PC3-MM2 to analyze surface CTLA-4. Briefly, mouse anti-human CTLA-4 mAb (BD Pharmingen TM, Europe) was used as the primary antibody to incubate the cell pellet for 30 minutes, and then incubated with Alexa at 4°C 488 donkey anti-mouse IgG (Invitrogen) was incubated as secondary antibody for an additional 30 minutes. In the LSR flow cytometer (BDPharmingen TM , Europe) to measure fluorescence intensity. Count at least 40,000 cells / sample. Usin...

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Abstract

The present invention relates to the fields of life sciences and medicine. Specifically, the invention relates to cancer therapies. More specifically, the present invention relates to oncolytic adenoviral vectors and cells and pharmaceutical compositions comprising said vectors. The present invention also relates to said vectors for treating cancer in a subject and a method of treating cancer in a subject. Furthermore, the present invention relates to methods of producing monoclonal anti-CTLA4 antibodies in a cell and increasing tumor specific immune response and apoptosis in a subject, as well as uses of the oncolytic adenoviral vectors for producing monoclonal anti-CTLA4 antibodies in a cell and increasing tumor specific immune response and apoptosis in a subject.

Description

field of invention [0001] The present invention relates to the fields of life science and medicine. In particular, the invention relates to cancer therapy. More specifically, the present invention relates to oncolytic adenoviral vectors as well as cells and pharmaceutical compositions comprising said vectors. The invention also relates to said vectors for use in treating cancer in a subject and methods for treating cancer in a subject. Furthermore, the present invention relates to methods of producing monoclonal anti-CTLA4 antibodies in cells and enhancing tumor-specific immune responses and apoptosis in a subject, and oncolytic adenoviral vectors for producing monoclonal anti-CTLA4 in cells Use of an antibody and enhancing tumor-specific immune response and apoptosis in a subject. Background of the invention [0002] Cancer can be treated with surgery, hormonal therapy, chemotherapy, radiation therapy, and / or other therapies, but in many cases, it is often characterized ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C07K16/28A61K35/761A61K39/00
CPCC07K16/2818C07K16/00C07K2317/21C12N2710/10343C07K2317/732A61K45/06C12N2810/6018C12N2710/10332A61K39/39558A61K35/761A61K39/00C12N15/8616A61K2039/505C07K2317/76C12N15/86C07K2317/73C12P21/00A61P35/00A61P37/04A61P43/00C12N15/861C07K16/28
Inventor J·迪亚斯V·切鲁洛A·海明基S·佩索宁
Owner ONCOS THERAPEUTICS
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