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Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
Published on: January 15, 2011
Production of bioactive soluble interleukin-15 in complex with interleukin-15 receptor alpha from a
David C Gaston1, Carl I Odom, Li Li
1Department of Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham, Birmingham, Alabama, United States of America ; School of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Abstract:
Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8(+) T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x10(7) plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.
Insights
Oncolytic herpes simplex viruses (oHSVs) engineered to express interleukin-15 (IL-15) and its receptor (IL-15Rα) effectively produced bioactive IL-15/IL-15Rα complexes. These complexes stimulated natural killer cells, showing potential for enhanced cancer immunotherapy.
Area of Science:
- Oncolytic virotherapy
- Immunotherapy
- Molecular engineering
Background:
- Oncolytic type-1 herpes simplex viruses (oHSVs) are engineered to selectively replicate in and kill cancer cells.
- oHSVs can be modified to express immunostimulatory molecules, such as cytokines, to enhance anti-tumor immune responses.
- Interleukin-15 (IL-15) is a cytokine that promotes natural killer (NK) and CD8(+) T cell-mediated cytotoxicity against cancer cells.
Purpose of the Study:
- To engineer an oHSV capable of producing bioactive IL-15.
- To investigate the role of IL-15 receptor α (IL-15Rα) in the production and bioactivity of IL-15 from oHSV.
Main Methods:
- Construction of two oHSVs: one expressing murine (m)IL-15 (J100) and another expressing both mIL-15 and mIL-15Rα (J100D).
- Assessment of oHSV replication competence, cytotoxicity in tumor cell lines, and production of mIL-15 and mIL-15Rα.
- Evaluation of the bioactivity of produced mIL-15/mIL-15Rα complexes on NK cell proliferation and cytotoxicity.
- Determination of the neurovirulence of engineered oHSVs in a mouse model.
Main Results:
- Both J100 and J100D oHSVs retained replication competence and direct cytotoxicity against permissive tumor cells.
- J100D demonstrated enhanced production of mIL-15 and secreted bioactive mIL-15/mIL-15Rα complexes, independent of oHSV permissiveness.
- The secreted mIL-15/mIL-15Rα complex from J100D stimulated NK cell proliferation and enhanced their cytotoxic activity against tumor cells.
- Both J100 and J100D were found to be aneurovirulent in vivo.
Conclusions:
- Co-expression of mIL-15Rα with mIL-15 in oHSV (J100D) enhances the production and secretion of bioactive IL-15/IL-15Rα complexes.
- The engineered oHSV J100D is capable of stimulating an anti-tumor immune response via NK cell activation.
- The aneurovirulence and enhanced IL-15 production profile of J100D make it a promising candidate for further investigation in cancer immunotherapy models.
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