Abstract 2502: Immunization with minicircle and mini-intronic DNA vectors induce LAG-3 expressing CD8+ T cells and inferior anti-tumor responses
Authors: Colluru Viswa Teja , McNeel Douglas G. 
Published: August 2015

BACKGROUND: Plasmid DNA-based gene delivery is a safe and economical vaccine modality that has demonstrated elicitation of robust cellular and humoral immunity and remarkable pre-clinical efficacy in multiple tumor models. In spite of this, DNA vaccines have been relatively unsuccessful in several human trials so far. One explanation for the difference in preclinical and clinical studies has been low or insufficient transgene expression from the plasmid vector, effectively reducing the antigen/body mass ratio in relatively large human subjects. Recent work in the gene therapy field has led to the discovery of a novel class of DNA vectors devoid of bacterial backbone sequences that leads to enhanced and sustained transgene expression in vitro and in vivo. In the following study we examined the potential of these vectors to induce enhanced immune responses compared to conventional plasmid vectors.

METHODS: Minicircle (DMC) and mini-intronic (MIP) plasmid vectors encoding the cancer testis antigen SSX-2 (synovial sarcoma breakpoint protein 2) were constructed from a conventional plasmid (pTVG-SSX2) and produced using standard methods. Sustained antigen expression was assessed following direct transfection of reporter cells after 2 or 7 days. The efficacy of these vectors as immunization vehicles was evaluated in HHDII-DRI transgenic mice (expressing human HLA-A2 and HLA-DR) by CD8 tetramer staining, IFNγ ELISPOT, intracellular cytokine staining, and in vivo anti-tumor response against syngeneic tumors expressing SSX2.

RESULTS: As expected, DMC and MIP vectors led to sustained and elevated levels of SSX2 protein in vitro when compared to standard plasmid DNA. Immunization with DMC-SSX2 led to a 3-fold increase in antigen-specific CD8+ T cells as measured by tetramer staining and with IFNγ release as measured by ELISPOT. However, these CD8+ T cells were less multifunctional as measured by IL2 and TNFα release using intracellular cytokine staining. This was associated with higher expression of LAG3 on SSX2-specific CD8 T cells. No significant changes in the expression of other regulatory markers (TIM3, PD1 or CD160) were noted. Immunization with a MIP-SSX2 construct similarly elicited higher numbers of antigen-specific LAG3+ CD8+ T cells and an inferior anti-tumor response compared to plasmid DNA.

CONCLUSION: Elevated and sustained antigen expression in vivo as mediated by minicircle and mini-intronic vectors led to the generation of greater frequencies of antigen-specific CD8+ T cells as compared to a standard plasmid vector. These T cells were, however, not multifunctional, expressed LAG3, and provided an inferior anti-tumor response compared to plasmid DNA. We suspect induction of tolerance to a DNA-encoded antigen might be mediated by sustained antigen presentation in a non-inflammatory context. Studies to evaluate this mechanism further are underway.

Citation Format: Viswa Teja Colluru, Douglas G. McNeel. Immunization with minicircle and mini-intronic DNA vectors induce LAG-3 expressing CD8+ T cells and inferior anti-tumor responses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2502. doi:10.1158/1538-7445.AM2015-2502