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Telomerase reverse transcriptase fusion protein, nucleotides encoding it, and uses thereof

A technology of fusion protein and reverse transcriptase, applied in the direction of fusion polypeptide, enzyme, transferase, etc.

Inactive Publication Date: 2009-09-02
IST DI RICERCHE DI BIOLOGIA MOLECOLARE P ANGELETTI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the development and commercialization of many vaccines has been hampered by difficulties associated with obtaining high-level expression of the desired immunogen in successfully transformed host organisms
The development of effective DNA-based vaccines has also been hampered by the inability to generate immune responses of sufficient magnitude in treated individuals to cause tumor regression under clinical conditions

Method used

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  • Telomerase reverse transcriptase fusion protein, nucleotides encoding it, and uses thereof
  • Telomerase reverse transcriptase fusion protein, nucleotides encoding it, and uses thereof
  • Telomerase reverse transcriptase fusion protein, nucleotides encoding it, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Construction of TERT fusion protein

[0132] In order to determine the relationship between the telomerase reverse transcriptase (TERT) antigen and the LTB subunit of Escherichia coli heat-labile enterotoxin ((Fingerut et al. ): Whether the fusion of 502-8(2004)) can enhance the immunogenicity of TERT alone, a vector encoding full-length telomerase reverse transcriptase with several modifications was constructed. First, the DNA sequence was codon Optimized to include codons preferred by human host cells. In addition, in order to ensure that the encoded antigen is safe for vaccine use, mutations were introduced into the TERT nucleotide sequence to inactivate the telomerase catalytic activity of the encoded protein. Specifically, Mutations D712A and V713I were added to the human TERT sequence, and mutations D702A and V703I were added to the mouse TERT sequence (Arai et al. Two independent regions of human telomerase reverse transcriptase are important for its oligomerizat...

Embodiment 2

[0136] Plasmid and adenoviral constructs

[0137] pV1JnsA / TPA-mTERT(AI)-LTBopt: Plasmid 041046pucKana containing the TPA-mTERT(AI)-LTB sequence was obtained from GENEART (Geneart GmbH, Regensburg, Germany). This plasmid was digested with BglII and SalI, and the resulting fragment was cloned into the BglII / SalI site in plasmid pV1JnsA (Montgomery et al., DNA Cell Biol., 12(9):777-83 (1993)).

[0138] pV1JnsA / mTERT(AI)opt: pV1JnsA / TPA-mTERT(AI)-LTB was digested with XbaI to remove the LTB coding sequence (between the two XbaI sites), resulting in construct pV1JnsA / TPA-mTERT with the stop codon after the last mTERT amino acid 3 amino acids (S; R; N) before the son. Removal of the TPA coding sequence was performed on pV1JnsA / TPA-mTERT(AI) by BamHI and EcoRV digestion. The TPA coding sequence was amplified with a sense primer (5'-G A T C T G A T G A T A T C G C C A C C A T G A C C A G A G C CC C C A G A T G-3'; SEQ ID NO: 15) and an antisense primer (5'-A G G G G G G A T CC G...

Embodiment 3

[0143] IFN-γ ELISPOT analysis

[0144] Mouse splenocytes secreting IFN-γ in an antigen-specific manner were detected using standard enzyme-linked immunospot (ELISPOT) assays (Miyahira et al. J Immunol Methods 181(1):45-54 (1995)). Ninety-six-well MAIP plates (Millipore Corp., Billerica, MA) were treated with 100 μl / well of purified rat anti-mouse IFN-γ (IgG1, clone R4-6A2) diluted to 2.5 μg / ml in sterile PBS. , Pharmingen) coated. After washing with PBS, blocking of the plate was performed with 200 μl / well of R10 medium at 37° C. for 2 hours.

[0145] Splenocytes were obtained by aseptically removing the spleen from euthanized mice and then disrupting the spleen by rubbing it on a wire mesh. Red blood cells were removed by osmotic lysis by adding 1 ml of 0.1×PBS to the cell pellet and vortexing for about 15 seconds. Then add 1 ml of 2×PBS and bring the volume to 4 ml with 1×PBS. Cells were pelleted by centrifugation at 1200 rpm for 10 minutes at RT, and the pellet was resu...

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Abstract

Polynucleotides encoding telomerase reverse transcriptase (TERT) fusion proteins are provided, the TERT fusion proteins comprising a TERT protein, or functional variant thereof, fused to a substantialportion of the B subunit of heat labile enterotoxin (LTB). TERT variants useful in TERT-LTB fusion proteins of the invention comprise mutations that function to eliminate telomerase catalytic activity. The polynucleotides of the present invention can elicit an immune response in a mammal, which, in preferred embodiments, is stronger than the immune response elicited by a wild-type TERT. TERT expression is commonly associated with the development of human carcinomas. The present invention provides compositions and methods to elicit or enhance immunity to the protein product expressed by the TERT tumor-associated antigen, wherein aberrant TERT expression is associated with a carcinoma or its development. This invention specifically provides adenoviral vector and plasmid constructs carryingpolynucleotides encoding TERT fusion proteins and TERT variants and discloses their use in vaccines and pharmaceutical compositions for preventing and treating cancer.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 60 / 851,183, filed October 12, 2006, the contents of which are hereby incorporated by reference in their entirety. field of invention [0003] The present invention relates generally to the treatment of cancer. More specifically, the present invention relates to polynucleotides encoding fusion proteins comprising at least a portion of the tumor-associated polypeptide telomerase reverse transcriptase (TERT). The invention also provides recombinant vectors and hosts comprising the polynucleotides, purified fusion proteins and methods of eliciting or enhancing an immune response against the protein product of the TERT gene using the compositions and molecules disclosed herein. Background of the invention [0004] Vaccination has become a standard method of preventing many infectious diseases. The application of vaccines to other diseases such as cancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435
CPCC12N9/1241C07K14/245C07K2319/00C12Y207/07049C07K2319/036A61P35/00C12N15/62C12N15/52C07K19/00C12N15/861
Inventor G·奇利伯托N·拉莫妮卡C·门努尼E·斯卡塞利
Owner IST DI RICERCHE DI BIOLOGIA MOLECOLARE P ANGELETTI
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