Fusion to Listeriolysin O and Delivery by Listeria monocytogenes Enhances the Immunogenicity of HER-2/neu and Reveals Subdominant Epitopes in the FVB/N Mouse

Abstract

Five overlapping fragments of rat HER-2/neu have been expressed in recombinant Listeria monocytogenes. Each fragment of HER-2/neu is secreted as a fusion protein with a truncated, nonhemolytic form of listeriolysin O (LLO). Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-EC3 overlap the extracellular domain of HER-2/neu, whereas Lm-LLO-IC1 and Lm-LLO-IC2 span the intracellular domain. All five strains controlled the growth of established NT-2 tumors, a rat HER-2/neu-expressing tumor line derived from a spontaneously arising mammary tumor in a FVB/N HER-2/neu-transgenic mouse. The antitumor effect of each of these vaccine constructs was abrogated by the in vivo depletion of CD8+ T cells, although only one known epitope has been defined previously and is present in Lm-LLO-EC2. Anti-HER-2/neu CTL responses were generated by each of the rLm vaccine constructs. With the use of a panel of 3T3 cell lines expressing overlapping fragments of HER-2/neu, regions of HER-2/neu with potential CD8+ T cell epitopes have been defined. DNA vaccines expressing either a fragment or full-length HER-2/neu were constructed in LLO-fused and non-LLO-fused forms. CTL analysis of the DNA vaccines revealed a broadening in the regions of HER-2/neu recognizable as targets when the target Ag is fused to LLO. These studies show the efficacy of L. monocytogenes-based HER-2/neu vaccines in a murine model of breast cancer and also that the immunogenicity of self-Ags can be increased by fusion to LLO and delivery by L. monocytogenes revealing subdominant epitopes.

Accumulation of CD8+ T Cells in Advanced-Stage Tumors and Delay of Disease Progression following Secondary Immunization against an Immunorecessive Epitope

“…Likewise, using the TRAMP mouse model of prostate cancer, Grossmann et al (16) reported the ability to recruit T CD8 against the SV40 Tag-V epitope, but did not assess the effect on tumor progression. Singh et al (54) recently described the discovery of subdominant T CD8 epitopes within the HER-2/neu Ag that could induce an antitumor response in mice that express HER-2/neu, but the ability of immunization to control spontaneous mammary tumors in these mice remains to be determined. Thus, to our knowledge, the effect of T CD8 specific for an immunorecessive tumor epitope on spontaneous tumor progression has not previously been explored.…”

Development of a live and highly attenuated Listeria monocytogenes-based vaccine for the treatment of Her2/neu-overexpressing cancers in human

“…The NT-2 tumor cell line, which expresses high levels of rat HER2/neu protein, was derived from a spontaneous mammary tumor in these mice 7 and grown as described previously. 4 DHFR-G8 (3T3/neu) cells were obtained from the American Type Culture Collection and were grown according to the American Type Culture Collection recommendations. The EMT6-Luc cell line was a generous gift from Dr John Ohlfest (University of Minnesota, MN) and was grown in C-RPMI medium.…”

“…3 In the first approaches to target the HER2/neu antigen using Listeria monocytogenes (Lm), Listeria-based vaccines expressing small fragments of the extra and intracellular domains of the rat and human HER2/neu protein were constructed. 4,5 We also generated a Listeria vaccine (Lm-LLO-ChHer2) using a chimeric protein by fusing two of the extracellular and one intracellular fragments of the human HER2/neu, including most of the known major histocompatibility complex (MHC) class I epitopes of the protein. 5 All of these vaccines were shown to be immunogenic and efficacious in regressing preestablished tumors in FVB/N mice and delay the onset of spontaneous mammary tumors in HER2/neu-expressing transgenic animals.…”

Mage-b vaccine delivered by recombinant Listeria monocytogenes is highly effective against breast cancer metastases

“…Mouse GM-CSF plasmid (CMV1-GM-CSF) was kindly provided by Dr Stephen Johnston (the Center for Innovations in Medicine, the Biodesign Institute at Arizona State University) (Chambers and Johnston, 2003). The listerial pGG-34 plasmid, expressing the positive regulatory factor A (prfA), was developed in the laboratory of Yvonne Paterson, University of Pennsylvania, PA, USA (Singh et al, 2005). The prfA-negative strain XFL-7 of LM (Gunn et al, 2001) has been used in this study.…”

“…Direct killing of tumour cells occurs through the function of CD8 T cells, but the killing may be enhanced through the activation of CD4 T cells (Hsieh et al, 1993). It has been shown that vaccine antigens delivered through LM are effective against primary tumours in animal models (Pan et al, 1999;Gunn et al, 2001;Singh et al, 2005). As discussed earlier, metastases, and not the primary tumour, contributes most to breast cancer morbidity and mortality.…”