Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Running title: In vivo antitumor Activity of a Recombinant IL-7/IL-15 Title: In vivo antitumor Activity of a Recombinant IL-7/IL-15 Hybrid Cytokine in Mice Yinhong Song1, 2, Yalan Liu1, Rong Hu1, Min Su1, Debra Rood1, and Laijun Lai1, 3 1 Department of Allied Health Sciences, 3 University of Connecticut Stem Cell Institute, University of Connecticut, Storrs, CT 2 Medical College, Three Gorges University, Yichang, China Keywords: cancer treatment, cytokines, IL-7, IL-15, mice Correspondence: Laijun Lai, M.D., Department of Allied Health Sciences, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269, USA. Phone: (860) 486-6073; Fax: (860) 486-0534; E-mail: [email protected] Potential Conflict of Interest: The authors declare that they have no conflict of interest. Financial Support: This work was partly supported by a grant from the Connecticut Biomedical Research Program (#2011-0145, to L. Lai). Word count: 4,591; total number of figures and tables: 6 1 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Both IL-7 and IL-15 have become important candidate immunomodulators for cancer treatment. However, IL-7 or IL-15 used alone suffers from shortcomings, such as short serum half-life and limited antitumor effect. We have cloned and expressed a recombinant (r) IL-7/IL-15 fusion protein in which IL-7 and IL-15 are linked by a flexible linker. We then compared the antitumor effect of rIL-7/IL-15 with the individual factors rIL-7 and/or rIL-15. We show here that rIL-7/IL-15 has a higher antitumor activity than the combination of the individual factors in both murine B16F10 melanoma and CT-26 colon cancer models. This was associated with a significant increase in tumor infiltration of T cells, DCs and NK cells and a decrease in regulatory T cells (Tregs). In addition, rIL-7/IL-15-treated DCs had higher expression of costimulatory molecules CD80 and CD86. The higher antitumor activity of rIL-7/IL-15 is likely due to its longer in vivo half-life and different effects on immune cells. Our results suggest that rIL-7/IL-15 may offer a new tool to enhance antitumor immunity and treat cancer. Introduction Both IL-7 and IL-15 are the common cytokine receptor γ–chain (γc) family cytokines. IL-7 plays a central role in the development and maintenance of T cells (1-5). The IL-7 receptor (R) consists of two subunits, the IL-7Rα and γc (1-5), the latter also being a component of the receptors for IL-2, IL-4, IL-9, IL-15 and IL-21. IL-7Rα is expressed by T 2 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cells and DCs, etc. (1-6). Several studies have shown that IL-7 has antitumor activity (7-12). For example, tumor cell lines that were transfected to produce rIL-7 locally reduced tumorigenicity in vivo, which was dependent on CD4+ or CD8+ T cells (7-9, 11). Local or systemic administration of rIL-7 also had antitumor effects (7, 10), especially when rIL-7 was combined with cancer vaccines (10, 12). IL-15 induces the differentiation and proliferation of T and NK cells, enhances the cytolytic activity of CD8+ T cells, and induces the maturation of DCs (13). The IL-15 receptor is composed of a unique α subunit (IL-15Rα), a β subunit (IL-2R/15Rβ) that is shared with the IL-2 receptor, and the γc. IL-15 can bind to IL-15Rβ via cis- or trans-presentation by IL-15Rα. IL-15Rβ is expressed by multiple lymphoid populations, such as T cells, DCs, NK cells, and NKT cells, etc. (14). In vivo administration of IL-15 has anti-tumor effects in several mouse tumor models (15-22); however, it has been shown that administration of IL-15 alone is not optimal (13). Various combination strategies have been explored to increase the efficacy of IL-15 immunotherapy, including coadministration of other cytokines or inhibitory antibodies against immune-suppression molecules (17-19, 22). These approaches produced greater anti-tumor responses than did IL-15 alone. Because both rIL-7 and rIL-15 have a low molecular weight, they undergo a rapid renal clearance in vivo, thereby having a short plasma half-life (23-26), which diminishes their in vivo antitumor effects (24, 25). Linking the coding sequences of IL-15 with other proteins has 3 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. increased the IL-15’s plasma half-life. Some of the fusion proteins also increase IL-15 signaling, thereby increasing its efficacy (24, 25, 27-29). We have previously described a single-chain rIL-7/HGFβ hybrid cytokine liking IL-7 and HGFβ by a flexible linker (30). The in vivo half-life of rIL-7/HGFβ was significantly longer than that of rIL-7 (31). In addition, rIL-7/HGFβ has different effects on immune cells (30-32), resulting in a higher antitumor activity (33), as compared with the individual factors rIL-7 and/or rHGFβ. Here we sought to determine whether a single-chain recombinant hybrid cytokine containing IL-7 and IL-15 might have a higher antitumor effect than the combination of the individual factors rIL-7 and rIL-15. Materials and Methods Animals and cell lines Murine B16F10 melanoma and CT-26 colon cancer cells were obtained from the American Type Culture Collection and the National Cancer Institute in 2008. American Type Culture Collection characterized the cells by using karyotyping and cytochrome c oxidase I testing. We passaged the cells for less than 4 months before storing them in liquid nitrogen. Before the cancer cells were injected into mice, they were cultured in Dulbecco Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS at 37℃ and 5% CO2 humidified air. Cell viability was assessed using a trypan blue exclusion dye method. Cells with a viability ＞95% were resuspended in PBS for injection. BALB/c, C57BL/6 mice, 4 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. homozygous C57BL/6 IFN-γ null mice (IFN-γ-/-), and homozygous TNF-α null mice (TNF-α-/-) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed, treated, and handled in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Connecticut. Construction, expression and purification of human single-chain IL-7/IL-15 hybrid cytokine. The human IL-7/IL-15 gene was constructed by adapting a protocol we previously used to construct the human IL-7/HGFα gene (32). Briefly, the human IL-7 cDNA was amplified with primers A [(that contains a secret sequence (SS)] and B, and the human IL-15 cDNA with primer C and D (Supplemental Table 1). The PCR products of IL-7 and IL-15 were combined and subjected to an additional round of PCR with primers A and D. Because primers B and C contained the linker sequence encoding (Gly4Ser)2, the IL-7 and IL-15 genes (IL-7/IL-15) were connected by a flexible linker after the overlap extension PCR. The IL-7/IL-15 gene was then cloned into a pOptiVEC mammalian expression vector (Invitrogen) (Figure 1A) that was then transfected into Chinese hamster ovary cell-derived DG44 cells (Invitrogen) to produce rIL-7/IL-15 protein. rIL-7/IL-15 protein was then purified from the supernatant of the transfected DG44 cells. Briefly, the supernatant was concentrated by a prep/scale–tangential flow filter cartridge with 10 kDa molecular weight cut-off (Millipore, Bedford, MA) and diafiltered into washing buffer. The sample was then applied to serially linked columns of CM and DEAE sepharose 5 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2016; DOI: 10.1158/1535-7163.MCT-16-0111 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. (GE Health Care Biosciences, Piscataway, NJ); after washing, the linked columns were separated. rIL-7/IL-15 protein was eluted from the DEAE column in the washing buffer containing NaCl gradient, and further purified by a gel filtration column (16/60 Sephacryl S-100 high resolution, GE). The purified protein was analyzed by SDS-PAGE and Western blotting using antibodies against human IL-7 and IL-15. For controls, we also cloned and expressed human IL-7 (32) or IL-15 (using primers E and D) gene individually, and purified rIL-7 and rIL-15 proteins from the expression system, respectively.