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GW0742 sale br Materials and methods br Results br Discussio
Materials and methods
Results
Discussion
It was previously shown that heterologous DNA vaccines composed of fused cDNA fragments encoding chimeric NH2-terminal human HER2 and COOH-terminal rat neu sequences stimulated stronger antibody responses and protective antitumor immunity than either HER2 or neu DNA vaccine in transgenic mice with HER2-specific self-immune tolerance [3], [20]. In this study, we constructed heterologous human/rat HER2-specific T cell vaccine HuRt-Texo by using ConA-stimulated polyclonal CD4+ T GW0742 sale with the uptake of EXO expressing HER2- and neu-specific pMHC complexes (EXOHuRt) released by DCHuRt expressing heterologous NH2-terminus HER2 and COOH-terminus neu fusion protein. For the first time, we demonstrate that heterologous HuRt-Texo vaccine, in comparison to homologous HER2-Texo vaccine, stimulates not only potent HER-2-specific antibody responses, which is consistent to previous reports [3], [20], but also more potent HER2-specific effector CTL responses, leading to enhanced therapeutic immunity against early stage-established HER2-expressing 4T1HER2 breast cancer in its lung metastasis or s.c. tumor form in wild-type BALB/c mice.
The Tg HER2 mice with HER2-specific self-immune tolerance [29] represent a useful animal model for assessing HER2-specific immunotherapies [35], [36]. We recently established a double Tg HLA-A2/HER2 mouse model by cross-breeding of Tg HER2 male with Tg HLA-A2 female mice. Thus, the double Tg HLA-A2/HER2 mice mimic HLA-A2+HER-2+ breast cancer patients with HER2-specific self immune tolerance. We demonstrated that HER2-TEXO vaccine stimulated HER2-specific CTL responses and partially induced protective immunity against transgene HLA-A2+HER2+ BL6-10A2/HER2 melanoma in double Tg HLA-A2/HER2 mice [24]. To assess whether heterologous human/rat HER2-specific T cell vaccine better circumvents HER2-specific immune tolerance, we immunized double Tg HLA-A2/HER2 mice with HuRt-Texo, followed by s.c. challenge of immunized mice with HLA-A2- and HER2-expressing BL6-10A2/HER2 tumor cells. We demonstrate that the HuRt-Texo vaccine triggers a strong protective immunity against BL6-10A2/HER2 tumors in all (8/8) tested double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance, which is significantly more efficient, in comparison to the protective immunity induced in only three from eight (3/8) mice by the HER2-Texo vaccination [24].
To assess the cytolytic effect of HuRt-TEXO-stimulated CTLs to trastuzumab-resistant breast cancer cells, we performed in vitro cytotoxicity assay, where HuRt-TEXO-stimulated CTLs and transgene HLA-A2-engineered trastuzumab-resistant breast cancer cell line BT474/A2 cells were used as effectors and targets, respectively. To examine the therapeutic effect of HuRt-TEXO-stimulated CTLs against trastuzumab-resistant breast cancer, we performed in vivo immunotherapeutic assay, in which BT474/A2 tumor-bearing athymic nude mice were adoptively transferred with HuRt-TEXO-stimulated CTLs. We found that HER2-specific CTLs derived from HuRt-TEXO-immunized HLA-A2/HER2 mice are not only cytolytic against BT474/A2 tumor cells in vitro, but also eradicate pre-established BT474/A2 tumors in athymic nude mice in vivo, revealing that HuRt-TEXO-stimulated CTLs are therapeutic against trastuzumab-resistant breast cancer.
CD8+ CTLs detecting HER2-specific pMHC-I complexes on HER2+ breast cancer cells are critical in protecting high-risk HER2+ breast cancer patients from cancer recurrence [37]. CD4+ helper T (Th) cells recognizing HER2-specific pMHC-II complexes provide important help for CD8+ CTL expansion and memory formation [38], [39]. HER2-specific MHC-II-restricted Th-epitopes would strengthen CTL response and memory derived from the MHC-I-restrictive HER2 peptide vaccine in HER2+ breast cancer patients [40]. Accumulating evidence also demonstrate that tumor-specific Th cells can also exhibit potent cytolytic effects against pMHC-II+ or pMHC-II- tumor cells [41], [42], [43], [44]. The P30 (FNNFTVSFWLRVPKVSASHLE) sequence of tetanus toxin is a strong heterologous CD4+ T helper epitope [45] capable of enhancing CTL responses via breaking self-immune tolerance in Tg mouse mammary tumor virus (MMTV)-c-neu and rat insulin promoter-mOVA mice, respectively [46], [47]. Vaccination with neu/P30 fusion protein completely inhibited neu+ breast cancer development in Tg FVNneuN mice [48]. We previously reported that vaccination of DCs engineered with neu transgene encoding P30 enhanced neu-specific protective immunity against neu-expressing mouse breast cancer Tg1-1 in Tg FVBNeuN mice with neu-specific self-immune tolerance [49]. In this study, we demonstrate that our heterologous human/rat HER2-specific T cell vaccine HuRt-TEXO stimulates stronger CD4+ T cell responses than the HER2-TEXO vaccine. We rationize that the potent CD4+ T cell responses in HuRt-TEXO-vaccinated mice are derived from the presence of heterologous Th epitopes within the COOH-neu481-590 portion of the HuRt-TEXO vaccine. It has been reported that eight HLA-DR-restricted HER2 Th epitopes were identified and clinically applied as a peptide vaccination to patients [50]. Among them, two peptides (HER262-76 and HER2605-619) are within the HER2 extracellular domain, while another six peptides located in the HER2 intracellular domain are mostly deleted in our heterologous human/rat HER2-specific T cell vaccine. We found that only one Th epitope neu605-619 (KPDLSYMPIWKYPDE) is within COOH-neu481-590 region of the HuRt fusion protein, but it contains a single amino acid F616Y mutation compared to HER2605-619 (KPDLSYMPIWKFPDE). Therefore, we suspect that the F616Y mutation in Th epitope may be critical in HuRt-TEXO’s enhanced circumvention of the HER2 tolerance. To test our assumption, construction of new HuRt-TEXO vaccine with F616Y mutation by recombinant DNA technology [27] followed by experiments designed to examine whether the F616Y mutation contributes to HuRt-TEXO vaccine’s effective circumvention of HER2 tolerance in double Tg HLA-A2/HER2 mice is underway in our laboratory.