Basic research of the OK-432-conjugated tumor vaccine

(1)OK-432-肿瘤疫苗的制备OK-432与肿瘤细胞(B16黑色素瘤细胞或Sq1979细胞)混合，并与GA偶联，以提高肿瘤细胞的抗原性。为了检测OK-432偶联肿瘤疫苗的安全性，进行了台盼蓝染色。超过80%的结合疫苗被台盼蓝染色，这表明大多数OK-432结合的肿瘤疫苗是不可行的。用不同浓度的赤霉素GA(0.2-0.0002%)免疫B16黑色素瘤细胞，在高浓度(0.02%以上)的条件下接合，对B16黑色素瘤细胞的存活率有强烈的抑制作用，180d后仍未观察到肿瘤。OK-432偶联瘤苗表面有抗OK-432抗体的FITC荧光强度染色，而未处理的肿瘤细胞未见染色。B16疫苗的三次免疫对B16黑色素瘤发病率的抑制作用最强，并伴随着…更多的死亡。KLN-205舌癌模型的制备为了分析OK-432偶联肿瘤疫苗的抗肿瘤作用机制，建立了小鼠舌癌实验模型。对两种鳞状细胞癌(KLN-205和Sq1979)进行了检测。在DBA/2小鼠舌癌模型中，KLN-205 2×10 5和5×10 5细胞组的成瘤率为100%。而在SQ-1979细胞模型中，5×105细胞组的成瘤率为100%，但肿瘤消退。因此，选择接种2×10~(-5)细胞/40μ的L KLN-205细胞作为舌癌的实验模型。(3)KLN-205疫苗的抗肿瘤作用分析为分析免疫小鼠接种KLN-205细胞后的变化。KLN205瘤苗组接种肿瘤后24小时，肿瘤细胞周围组织切片可见明显的浸润性淋巴细胞。对照组可见散在分布的淋巴细胞，未见浸润性淋巴细胞。(4)KLN205疫苗免疫小鼠的细胞杀伤活性，KLN205疫苗免疫的小鼠脾细胞对KLN-205细胞的杀伤活性高于其他疫苗免疫的小鼠。但经赤霉素处理的KLN-205、经赤霉素A处理的OK-432和单独处理的OK-432表现出与对照几乎相同的百分率特异性裂解。这些发现表明，两者(肿瘤细胞和OK-432)的结合对于使用基于细胞的疫苗进行癌症免疫治疗具有重要意义。较少

(1)Preparation of the OK-432-conjugated tumor vaccinesOK-432 was mixed with tumor cells (B16 melanoma cells or Sq1979 cells), and conjugated with the addition of GA to increase the antigenicity of tumor cells. To examine the safety of the OK-432-conjugated tumor vaccines, a trypan blue staining was performed. Over 80 % of the conjugated vaccines were stained with trypan blue, indicating that the majority of OK-432-conjugated tumor vaccines were not viable. When tumor cells treated with various concentrations of GA (0.2-0.0002 %) were immunized, conjugation under condition of high concentrations of over 0.02 % GA inhibited strongly viability of B16 melanoma cells, so that tumor was not observed up to 180 days. The surface of OK-432-conjugated tumor vaccines was strongly stained with FITC emission of anti-OK-432 antibodies, while untreated tumor cells were not stained. Three immunizations with the B16-vaccines showed the strongest suppression of the incidence of B16 melanoma and associat … More ed mortality. The only immunizations with the Sq1979-vaccines induce anti-Sq1979 specific effects, and the B16-vaccines elicit no cross-reactive antitumor effects.(2) Preparation of KLN-205 tongue cancer modelTo analyze the mechanisms of antitumor effects by the OK-432-conjugated tumor vaccines I developed the experimental murine tongue cancer model. Two kinds of squamous cell carcinoma (KLN-205 and Sq1979 cells) were examined. In the DBA/2 mice tongue cancer model, the oncogenetic rate was 100 % in the groups of 2×10^5 and 5×10^5 cells of KLN-205. On the other hand, the oncogenetic rate of the 5×10^5 cell group was 100 % in the Sq-1979 cell model, but tumor regression was observed. Therefore, inoculation of 2×10^5 cells / 40 μl KLN-205 cells was selected as an appropriate experimental tongue cancer model.(3) Analysis of antitumor effects induced by the KLN205-vaccinesTo analyze the changes of the immunized mice after inoculation of KLN-205 cells, I carried out histological examination. Infiltrating lymphocytes were markedly observed around the tumor cells with histological section at 24h after tumor inoculation in the KLN205-vaccine group. On the other hand, scattering lymphocytes were shown and no infiltrating lymphocytes could be observed in the control group. These results indicate that immunized mice obtained immunological memory for KLN-205 cells.(4) The KLN205-vaccines elicit cytolytic activityIn the cytolytic assay, splenocytes from mice immunized with the KLN205-vaccines indicate a greater cytolytic response than that from mice immunized with the others. But KLN-205 treated with GA, OK-432 treated with GA and OK-432 alone showed almost the same level of precent specific lysis as control. These findings suggest the conjugation of both (tumor cells and OK-432) is of importance concerning cancer immunotherapy using a cell-based vaccine. Less