Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model

Abstract

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.

Fig 1. Vector schematics.

The hyperactive (m7pB) piggyBac transposase was used in combination with various transposons for mIL-12 and/or reporter gene (venus or luciferase) expression in vitro or in vivo. CMV, cytomegalovirus immediate early enhancer/promoter; piggyBac, transposase; pA, SV40 polyadenylation signal; mIL-12, murine IL-12; 2A, 2A sequence; venus, reporter gene; effLuc, enhanced luciferase reporter gene; stop, stop codon; IRES, internal ribosomal entry site; Thy1.1, mouse Thy1.1 antigen; WPRE, woodchuck hepatitis post-transcriptional regulatory element; eGFP, enhanced green fluorescent protein.

Fig 2. Functional expression of IL-12 from a piggyBac transposon.

A, HeLa cells were transfected with the pT-IL12-2A-venus transposon. DAPI (4',6-diamidino-2-phenylindole) stain was utilized to visualize cell nuclei (top) and immunofluorescence of the venus reporter gene was used to visualize protein expression (bottom). Shown is a representative of 3 independent experiments. Culture media from these cells was analyzed for mIL-12 production resulting in 31 ± 5pg/μl of mIL-12 (N = 3, ± SEM). B, B16 melanoma cells were stably transfected with pT-mIL12 in the presence of pCMV-m7pB. 5 X 10⁵ B16 cells were implanted into mice on the hind quarter. The ability of mIL-12 expressing B16 cells to affect the growth of contralaterally implanted B16 cells was compared to that of untransfected cells. mIL-12 expressing B16 cells inhibited the contralateral growth of B16 cell in vivo.

Fig 3. piggyBac transposon modification of mouse splenocytes.

A, Splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1 using the Neon transfection system and transfection efficiency was evaluated via flow cytometry using antibodies directed against the Thy1.1 antigen at day 1 and day 7 post transfection. Shown is a representative of 3 independent experiments. B, mouse splenocytes could be cultured short term exhibiting cell growth. Shown is a representative of 3 independent experiments. Arrows indicate a split to 5 X 10⁶ cells for continued growth.

Fig 4. Homing of piggyBac-modified mouse splenocytes to tumor sites in vivo.

OT-I mouse splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1. 5 X 10⁵ B16/OVA cells into the flank of C57Bl6 mice (day –8). piggyBac-modified splenocytes were adoptively transferred via tail vein injection on day 0 and day +8. Localization of infused splenocytes was visualized via in vivo imaging of luciferase expression on day +11. Show are 3 of 6 representative animals.

Fig 5. IL-12 transfected OT-I cells produce IL-12 and produce IFNγ when co-cultured with B16/OVA cells.

A, OT-I splenocytes were transfected with pT-eGFP (control) or pT-mIL12 and co-cultured with B16 or B16/OVA cells. Flow cytometry confirmed the presence of eGFP expressing CD8 positive OT-I cells at the end of the co-culture. Shown is a representative of 3 independent experiments. B, cytometric bead analysis was used to measure mIL-12 (au, arbitrary units) in the media derived from the co-culture. *, p<0.05 comparing mIL-12 groups to eGFP. C, cytometric bead analysis was used to measure INFg production from transfected OT-I cells in the presence of B16 or B16/OVA cells. *, p<0.05 comparing B16/OVA groups to B16 (without OVA).

Fig 6. IL-12 piggyBac-modified mouse splenocytes exhibit anti-tumor activity in vivo.

A, 5 X 10⁵ B16/OVA cells were transplanted into the flank of C57Bl6 mice.OT-I splenocytes modified with pT-mIL12 and pCMV-m7pB (compared to pCMV-m7pB alone) were adoptively transferred on day 0 and day 8 and tumor growth was monitored in vivo via caliper measurement of tumor diameter. *, p<0.05 using the student’s T test on the given day of comparison. OT-I splenocytes modified with pT-mIL12 slowed tumor growth in vivo. B, Adoptive transfer of piggyBac modified OT-1 splenocytes also improved mouse survival in the B16 melanoma model. The Mantel-Cox test exhibited a statistically different survival between the two groups, N = 10.