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- Materials and Methods
Recently, dendritic cells (DC) transfected with tumor RNA have been used as a cancer vaccine. The efficacy of a cancer vaccine using DC transfected tumor RNA was examined. Of particular interest was whether a vaccine using DC transfected with recrudescent tumor RNA is effective for the treatment of a regrowing tumor after prior immunotherapy. In addition, the usefulness of co-transfection of granulocyte macrophage colony-stimulating factor (GM-CSF) mRNA to augment the DC vaccine was examined. CT26 tumor-bearing mice were immunized by s.c. injection with DC transfected with CT26 mRNA (DC-CT26). The cytotoxic activity against CT26 in mice immunized with DC-CT26 was significantly higher than that in the control group (P < 0.001) and was augmented by GM-CSF mRNA co-transfection (P < 0.05), resulting in remarkable therapeutic efficacy in CT26 s.c. tumor models. Cytotoxic T lymphocytes induced by the vaccination using DC transfected with mRNA from the recrudescent tumor showed a potent cytotoxicity against the recrudescent CT26 tumor cells, which was significantly higher than the cytotoxicity induced by the vaccination using DC-CT26 (P < 0.05). In addition, in a recrudescent tumor model, this vaccination suppressed the regrowing s.c. tumors, and was augmented by GM-CSF mRNA co-transfection (P < 0.05). These results suggested that vaccination therapy using DC simultaneously transfected with whole tumor RNA and GM-CSF mRNA could generate therapeutic immune responses even against recrudescent tumor after prior vaccination. (Cancer Sci 2008; 99: 407–413)
Immunotherapy against cancer has been extensively studied. It has recently been reported that immunotherapy using antigen-loaded dendritic cells (DC) (sipuleucel-T) has provided a survival advantage against asymptomatic hormone-refractory metastatic prostate carcinoma patients for the first time.(1) However, most immunotherapy using single-antigen-loaded DC is insufficient because of heterogeneity of tumors. Another approach, utilizing total tumor proteins or tumor associated antigen (TAA) genes as a source of antigen have been developed; for example, a vaccine using DC loaded with tumor lysates,(2) proteins(3) or DNA.(4,5) Recently, RNA has also been used for tumor vaccination.(6–8) Boczkowski et al. reported the first functional data that DC transfected with mRNA-encoding specific tumor antigens or total tumor RNA derived from tumor cells elicited potent specific cytotoxic T lymphocytes (CTL) responses against tumors.(6) The strategy of using RNA has several potential advantages. First, RNA can be effectively amplified from a very small number of cells; therefore, unlike tumor-extract vaccines, a small amount of tumor tissue is sufficient to prepare the material for vaccination.(7) Second, the tumor antigen need not be identified and the presence of multiple tumor antigens reduces the risk of antigen-negative escape mutants. Furthermore, it is not necessary to elucidate the molecular nature of the putative tumor antigens. Third, unlike DNA-based vaccines, there is little danger of incorporation of RNA sequences into the host genome. On the other hand, there are several drawbacks to the use of RNA for vaccination. RNA is unstable, and it seems to be difficult to maintain its quality for clinical application; and, moreover, there is only a low level of protein expression caused by RNA transfection. However, in spite of this, several clinical trials of cancer vaccines using DC transfected with tumor derived RNA showed clinical feasibility and safety in the treatment of patients and also showed that obvious antitumor immune responses were induced.(9–11)
Another remarkable advantage of RNA-loaded DC-based vaccines is the ability to deal with the recrudescent tumor, even in the same patient, because RNA can be freshly prepared from the recurrent tumor via biopsy. It is very important to establish the method of enhancing the antitumor effect of tumor-RNA-loaded DC-based vaccines, considering future clinical application. The functions of DC are affected by several immunostimulatory cytokines within the local tissue environment.(12) In particular, granulocyte macrophage colony-stimulating factor (GM-CSF) is a potent stimulator of DC.(13) Several investigators have reported that GM-CSF gene-transduced tumor vaccine results in efficient tumor suppression and survival benefit in mouse models.(14,15) Furthermore, DC adenovirally transduced with the whole TAA gene and GM-CSF gene strengthened T-cell responses, especially their migratory capacity for draining the lymph node by CC chemokine receptor 7 (CCR7) expression.(16) Therefore, it is possible that GM-CSF is a good candidate for the enhancement of this vaccine therapy. This study was designed to investigate whether immunotherapy using DC transfected with tumor RNA is useful for the treatment of s.c. tumors. Of particular interest was to examine whether DC transfected with recrudescent tumor RNA are effective for the treatment of a regrowing tumor after vaccination using DC transfected with primary tumor RNA. Furthermore, the augmentation effect of co-transfection of DC with GM-CSF mRNA on this vaccine strategy was investigated.
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- Materials and Methods
The present study demonstrated that vaccination therapy using DC transfected with tumor RNA could induce an antitumor immune response as Boczkowski et al. have reported.(6) The vaccination using DC transfected with CT26 mRNA elicited tumor-specific CTL, because they selectively lyzed CT26 tumor cells and AH-1 pulsed Meth-A. However, the cytotoxicity of these CTL was not sufficient to achieve an in vivo antitumor effect against CT26 s.c. tumors. Therefore, a mechanism for the augmentation of the antitumor effect is necessary. GM-CSF mRNA was employed for the enhancement of this cancer vaccine therapy. GM-CSF is a potent stimulator of DC and GM-CSF gene transduction enhances the capacity of DC to induce a primary immune response.(13,22) GM-CSF plays an important role in the maturation, function and migration capacity of DC and mediates homing to lymphoid organs in response to the cognate ligands CCL19 and CCL21.(23) In addition, it has recently been reported that GM-CSF mRNA encapsulated in cationic liposomes enhances immunological responses induced by the vaccination with tumor associated antigens.(24) Our previous studies demonstrated that a vaccination using DC co-transduced with the TAA gene and the GM-CSF gene elicited potent therapeutic immunity in s.c. tumors in mouse models,(16,25) and also showed that the co-transduction of the GM-CSF gene enhanced the migratory capacity of DC for draining the lymph node by upregulation of CCR7 expression.(16) In addition, GM-CSF has other important functions for DC. We also showed that the lifespan of DC transfected with the GM-CSF gene was prolonged, and GM-CSF might protect DC from apoptosis induced by tumor-derived transforming growth factor (TGF)-β-1 in the regional lymph nodes.(26)
In the present study, DC were simultaneously transfected with CT26 mRNA and the GM-CSF mRNA, and it was confirmed that GM-CSF was really secreted by transfected DC. These findings showed that specific CTL activity against CT26 in the spleen cells from mice immunized with DC transfected with CT26 was enhanced by co-transfection of DC with the GM-CSF mRNA. In addition, the antitumor effect of the vaccination was enhanced by co-transfection with GM-CSF mRNA in CT26 tumor models. However, this augmenting efficiency by co-transfection of GM-CSF mRNA was not as potent as that of adenoviral GM-CSF gene transduction shown in a previous study.(16) The reason for this is because the amount of GM-CSF produced by DC transfected with GM-CSF mRNA was reported to be only approximately 1% of the optimal dose produced by DC adenovirally transduced with the GM-CSF gene in the previous study.(16)
One of the major advantages in using mRNA for TAA gene transfection into DC is to deal with residual tumors or recrudescent tumors. The critical obstacle of cancer immunotherapy is that tumors evade immune surveillance via several mechanisms, such as loss or downregulation of human leukocyte antigen (HLA) class I,(27) antigen alternation or loss of tumor antigens,(28) influence of immunosuppressive cytokines,(29) and suppressor T cells.(30) It is well documented that tumor antigen expression is heterogeneous, even within the same tumor. Decreased antigen expression has also been found in residual tumors after peptide vaccination.(31,32) In the clinical studies of peptide vaccine for melanoma, the expression of tumor antigen has been found to obviously decrease in residual tumors after peptide vaccination.(33) In the animal experiments, antigenic drift has been reported to cause tumor evasion.(34) Tumor escape variants probably emerge after treatment with increasingly effective immunotherapy.(35) It seems likely that, as T-cell-based tumor immunotherapy becomes stronger, escape mechanisms such as antigen loss are likely to become more prominent. Tumors can alter their antigen expression qualitatively through the mutation of antigenic epitopes. In fact, in the present study, CTL induced by the vaccination using DC-transfected mRNA from the recrudescent tumor showed a potent cytotoxicity against the recrudescent tumor cells, which was significantly higher than the cytotoxicity induced by the vaccination using DC transfected with the primary tumor. These data were consistent with the therapeutic effect observed in s.c. tumor models. We speculated that the CT26 tumor altered the antigenicity after the vaccination using DC transfected with the primary CT26 tumor, and then a subpopulation of CT26 tumor cells survived and evaded immune surveillance, and therefore the vaccination using DC transfected with the recrudescent CT26 tumor was more effective than the vaccination using DC transfected with the primary CT26 tumor. However, further experiments comparing the gene expression profiles between CT26 and CT26R are necessary to assess the difference between CT26 and CT26R in terms of antigenicity.
Considering the clinical application, it is not possible to obtain a large amount of tumor tissue from patients with recurrent tumors. Therefore, a vaccine using DC pulsed with tumor lysate is not feasible in such situations. However, there is no such problem if tumor mRNA is used, because it can be amplified without loss of function.(7) There is another advantage in using mRNA for genetic modification of DC: it is easy to transfect several cytokines at the same time. In the present study, DC were transfected with tumor mRNA together with GM-CSF mRNA to augment the vaccine efficiency. Moreover, co-transfection of other cytokine genes such as interleukin (IL)-12 would be expected to further modify DC function.(25) However, this vaccine strategy may not be expected to reduce a grown tumor, considering reports of previous clinical studies.(9–11) Therefore, this strategy should be used to obtain a maximum benefit. For example, a cancer vaccine using DC transfected with mRNA extracted from surgical specimens may be useful as an individualized adjuvant immunotherapy to prevent metastasis and recurrences after surgery. Once the tumor has recurred, mRNA could be extracted from biopsy specimens of the recurrent tumor and amplified. Then, it could be transfected into DC together with cytokine mRNA such as GM-CSF and so on. Vaccination using these DC may be useful for further immunotherapy.
In conclusion, the vaccine strategy using DC transfected simultaneously with tumor mRNA and GM-CSF mRNA resulted in the generation of efficient therapeutic immune responses, even against recrudescent tumors. Therefore, this therapeutic strategy is promising for clinical application as an effective cancer vaccine both in an adjuvant setting after a surgical resection and in the treatment of recurrent tumors.