The genes encoding immunostimulatory molecules with anti-tumoric effects include IL-2 (interleukin-2), IL-4, IL-7, IL-12.

□Efficient coexpressing of IL-12 and IL-23 , □Higher expression levels of the IL-12 and IL-23 proteins., □Excellent anti-tumor effects

l  Technology Platform

The core technology of Yonsei University is to provide a method for coexpressing IL-12 and IL-23, which comprises the steps of: (a) preparing vectors comprising monocistronic expression constructs of each of nucleotide sequences encoding the p35 subunit, the p40 subunit and the p19 subunit, or preparing a vector comprising a polycistronic expression construct of nucleotide sequences encoding the p35 subunit, the p40 subunit and the p19 subunit; (b)  transforming the expression          constructs into a host cell; and (c) culturing the transformed host cell to obtain IL-12 and IL-23, a vector for coexpressing        IL-12 and IL-23, and a pharmaceutical anti-tumor composition comprising the vectors.

Background and unmet needs: Because most cancers were diagnosed after the occurrence of metastasis into   other organs or tissues without apparent symptoms, surgical operations, radiotherapy and anti-tumor chemotherapy in use as  current therapies have exhibited many limitations.

Immunotherapy of cancer is aimed to specifically remove cancer cells by boosting the innate immune system. Due to the rapid development of genetic engineering and molecular immunology, immune cells and their action mechanisms         inducing anti-tumor immune response were revealed, resulting in new turning point in the field of anti-tumor immunotherapy. Much interests and researches have been progressed up to date.

In spite of the rapid development and many efforts, immunotherapy has revealed many limitations to treat cancers. Because cancer cells could avoid ingeniously the immune system through various strategies and escape from activated anti-tumor immune response, they could proliferate continuously in the event. To be free from these limitations, the immune response against tumors has been induced by introducing immunostimulatory cytokine genes directly into cancer cells for producing and secreting cytokines in cancer cells per se, such that cancer cells are specifically removed.

 

Discovery and Achievements: Dr. Chae Ok, Yun have made intensive studies to coexpress IL-12 and IL-23 having anti-tumor effect in much higher efficiency and to develop gene therapeutics with enhanced anti-tumor effect. As results, She has discovered a suitable combination of genes encoding the subunits of IL-12 and IL-23 could permit to coexpress efficiently IL-12 and IL-23 and to exhibit enhanced anti-tumor effects.

 

For applying effectively adenoviral gene therapy for cancers to practical therapeutics,  the development of adenovirus having both the specificity to kill selectively cancer cells without side effects on surrounding normal cells and the capability to kill effectively cancer cells is indispensable. However, because 1^st replication-incompetent adenoviruses in which the E1A gene essential for adenovirus replication was deleted, exhibited the infectiveness in only the first generation, they induced anti-tumor activity solely in infected cells and a very small number of surrounding cells, rendering them to have serious problems in clinical applications.

To overcome such problems, Yonsei University reported both cancer-specific proliferation and oncolytic effects through the development of the E1B 55 kDa-deleted tumor-specific adenovirus, YKL-1 (Ad-ΔE1B55), which could be proliferated selectively in cancer cells lacking functional p53, tumor-inhibitory protein. The oncolytic adenovirus which could proliferate selectively in the cancer cells and kill them, exhibited therapeutic effects in primarily infected cells and in turn proliferated adenoviruses infected and killed surrounding tumor cells. These features lead to dramatic cancer treatment efficacy with little or no adverse effects due to their incompetence of replication in normal cell. However, the proliferation of YKL-1 adenovirus was restricted relatively as compared with wild type (WT) adenovirus, and its cytotoxicity was reduced significantly, resulting in low anti-tumor effects²⁵.

To augment such lower cell-killing ability of YKL-1 adenovirus, Yonsei University had developed and reported in vivo and in vitro excellent anti-tumor effects of Ad-ΔB7 adenovirus showing enhanced cell-killing potential through the deletion of E1B 19 kDa region and significantly improved oncolytic activity through substituting Gly (G) for Glu (E) in 45 position within CR1 region and seven GGGGGGG for seven DLTCHEA in CR2 region. That is, the Ad-ΔB7 adenovirus has not only enhanced oncolytic activity against cancer cells due to increased apoptosis-inducing potential by the deletion of the E1B 19 kDa region to inhibit apoptosis, but also improved oncolytic selectivity by the deletion of the Rb-binding region in the E1A gene and substitution with Gly residue to enable it to replicate only in Rb-mutated cancer cells.

 

Method for coexpressing IL-12 (interleukin-12) and IL-23 (interleukin-23) comprises the steps of: (a) preparing vectors comprising monocistronic expression constructs of each of nucleotide sequences encoding the p35 subunit, the p40 subunit and the p19 subunit, or preparing a vector comprising a polycistronic expression construct of nucleotide sequences encoding the p35 subunit, the p40 subunit and the p19 subunit; (b) transforming the expression constructs into a host cell; and (c) culturing the transformed host cell to obtain IL-12 and IL-23.

 

Anti-tumor effects : To determine anti-tumor effect of each oncolytic adenoviruses, which was Ad-ΔB7/IL12 and Ad-ΔB7/IL23 expressing each IL-12 and IL-23, or Ad-ΔB7/IL23-p35 expressing both IL-12 and IL-23, B16-F10 murine melanoma cell line was injected subcutaneous on the abdomen of male C57BL/6 mice and mice injected with PBS or Ad-ΔB7 were all died each at 11 or 12 days after injection, but mice with Ad-ΔB7/IL23 were all died at 18 days, demonstrating that the survival rate was increased in the Ad-ΔB7/IL23 than in PBS or Ad-ΔB7 as a control.

To determine the occurrence frequency of tumor-specific immune cells secreting IFN-γ, IFN-γ ELISpot analysis was performed, and 6 × 10⁵ splenocytes extracted from murine spleen injected with each virus. It was observed especially that the number of brown spot in mouse injected with Ad-ΔB7/IL23-p35 was increased greater than with Ad-ΔB7, Ad-ΔB7/IL12.

To examine the changes within tumor tissue when IL-12 and IL-23 were expressed, lymphocytes infiltrated into tumors were investigated. As a result of H & E staining about tumor tissue injected with each viruses or PBS, it was observed  that

lymphocytes were not infiltrated into tumor tissues in the sample injected with PBS, but infiltrated into tumor tissues around in the samples injected with Ad-ΔB7/IL12 or Ad-ΔB7/IL23. Additionally, it was confirmed that in the samples of tumor tissues injected with Ad-ΔB7/IL23-p35 as compared with Ad-ΔB7/IL12 or Ad-ΔB7/IL23, many lymphocytes were penetrated notably not only surrounding, but inside tumor tissues, and most tumor cells were died.

Early Stage

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