Oncolytic virus (oncolytic immunotherapy) capable of effectively treating even metastatic cancer while ensuring safety, with expression control system providing optimal expression level of mounted immunogenic gene

A technology of tumor dissolution and immune induction, applied in the direction of non-active ingredient medical preparations, medical preparations containing active ingredients, viruses, etc., can solve difficult, difficult gene recombination, efficient and standardized production of conditional replication adenovirus Technology does not exist and other issues

Pending Publication Date: 2020-08-14
KAGOSHIMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although adenovirus is a medium-sized virus with a genome size of 30-40kb, genetic recombination is not easy. Unlike non-proliferating adenoviruses, there is no efficient and standardized production technology for conditionally replicating adenoviruses
Therefore, it is difficult to develop m-CRAs with high degree of gene recombination at multiple positions, and then to make multiple candidate m-CRAs and conduct screening comparative experiments.

Method used

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  • Oncolytic virus (oncolytic immunotherapy) capable of effectively treating even metastatic cancer while ensuring safety, with expression control system providing optimal expression level of mounted immunogenic gene
  • Oncolytic virus (oncolytic immunotherapy) capable of effectively treating even metastatic cancer while ensuring safety, with expression control system providing optimal expression level of mounted immunogenic gene
  • Oncolytic virus (oncolytic immunotherapy) capable of effectively treating even metastatic cancer while ensuring safety, with expression control system providing optimal expression level of mounted immunogenic gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] (Example 1) GM-CSF expresses the structure of Surv.m-CRA

[0116] The Surv.m-CRA with the survivin promoter integrated upstream of the early gene E1A necessary for adenovirus growth was inserted. The promoters of the E2F promoter, RSV promoter, and CA promoter were linked to the mouse GM-CSF cDNA. And the expression box that obtains, thereby constructs following three kinds of GM-CSF expression Surv.m-CRA ( figure 1 ). The construction of adenovirus was carried out based on the method described in GeneTherapy (2005, 12:1385-1393) by Nagano et al.

[0117] (1) Production of P1+3 plasmid

[0118] As the first stage of production, P1+3 was produced from the growth control plasmid P1 and the adenovirus backbone plasmid P3 (pAd.HM4, pAd.HM10; Mizuguchi and Kay, Hum. Gene Ther. 1999) by restriction enzyme treatment.

[0119] The adenovirus backbone plasmid P3 is loaded with human adenovirus type 5 genomic DNA, but lacks the E1 gene region required for virus propagation. I...

Embodiment 2

[0148] (Example 2) Infection efficiency of adenovirus in hamster-derived cells

[0149] The infection efficiency of adenovirus in hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) was examined.

[0150] HaK cells derived from Syrian hamster kidney tumor were provided by Prof. M. Wold, Saint Louis University School of Medicine. Use DMEM (Dulbecco's Modified Eagle Medium; Nacalai Tesque) containing 10% fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (Nacalai Tesque) at 37° C. , 5%CO 2 cultured under conditions. HaP-T1 cells derived from Syrian hamster pancreatic tumor were purchased from RIKEN BRC Cell Bank (RBRC-RCB0411). With 10% FBS, 1% non-essential amino acids (NEAA; SIGMA), 1 mM sodium pyruvate (Thermo Fisher Scientific) and 1% penicillin-streptomycin MEM (Minimum Essential Medium; SIGMA) at 37°C, 5% CO 2cultured under conditions. BHK-21 cells derived from Syrian suckling hamster kidney were purchased from JCRB Cell Bank (JCRB90...

Embodiment 3

[0153] (Example 3) Promoter activity in hamster-derived cells

[0154] The promoter activities of E2F, RSV, and survivin in hamster-derived cancer cells (HaK and HaP-T1) and normal cells (BHK-21) were investigated. On the day of infection, the cells seeded in the 6-well plate the day before were counted, and the result was: HaK was 1.29×10 6 cells / well, HaP-T1 is 1.0×10 6 cells / well, BHK-21 is 1.7×10 6 cells / well. Next, Ad.dE1.3 (control) in which genes E1 and E3 important for virus growth have been deleted, and Ad.E2Fp expressing the β-galactosidase gene (LacZ) under the control of each promoter -LacZ, Ad.RSVp-LacZ and Ad.Survp-LacZ were infected for 1 hour under the condition of MOI30, and cultured for 48 hours. Then, the β-galactosidase activity in the cell lysate was examined using the Beta-Glo (registered trademark) Assay System (Promega). Experiments were performed in groups of three wells under each condition, and the data were expressed as mean ± standard error. ...

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Abstract

A purpose of the present invention is to develop an immuno-viral therapy vector that has an optimal therapeutic effect while ensuring a high degree of safety, on the basis of the novel concept of finding the optimal expression level of a therapeutic gene to bring about the maximum therapeutic effect with no side effects. The present invention provides an oncolytic virus or similar that is characterized by having an immunogenic gene that is functionally linked downstream of an E2F promoter or a promoter demonstrating the same activity as an E2F promoter, wherein a promoter of nucleic acids coding for at least one factor that is essential for virus replication or assembly is substituted with a promoter of a factor for which expression is organ-specifically enhanced, or a promoter of a factorfor which expression is cancer cell-specifically enhanced.

Description

technical field [0001] The present invention relates to the field of cancer therapy, so-called oncolytic immunotherapy, using oncolytic viruses loaded with immune-inducing genes. Background technique [0002] Oncolytic immunotherapy is an immunotherapy that loads an immune-inducing gene into an oncolytic virus, which is a genetically recombinant virus that specifically proliferates in cancer cells and exhibits a killing effect. , which is expected to be the most powerful candidate for innovative cancer treatment drugs in the world (Non-Patent Document 1-Non-Patent Document 3), at the end of 2015, Europe and the United States approved the T-Vec gene loaded with cytokine genes (Amgen's T-Vec ) medicines as First-in-class (epoch-making medicines). [0003] In the early 1990s of gene therapy, the inventors of the present invention were the first in the world to develop immune gene therapy by introducing immune genes using non-proliferative viral vectors (Non-Patent Documents 4 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K35/761A61K47/55A61K48/00A61P35/00A61P35/04A61P37/04A61P43/00C12N15/861
CPCA61K35/761A61P35/00A61P35/04A61P37/04A61P43/00C12N15/86C12N2710/10343C12N2830/008C12N2710/10332A61K48/005C07K14/52Y02A50/30C12N15/861
Inventor 小戝健一郎伊地知畅广
Owner KAGOSHIMA UNIV
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