Basic research of the OK-432-conjugated tumor vaccine

OK-432结合肿瘤疫苗的基础研究

• 批准号: 12671835
• 负责人: BUKAWA Hiroki
• 金额: $ 2.18万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671835/
• 关键词: OK-432; Tumor vaccine; conjugation; KLN-205; CTL; アポトーシス; B16; Sq-1979

(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

将OK-432与肿瘤细胞（B16黑色素瘤细胞或Sq 1979细胞）混合，并通过添加GA进行缀合以增加肿瘤细胞的抗原性。为了检查OK-432缀合的肿瘤疫苗的安全性，进行台盼蓝染色。超过80%的缀合疫苗用台盼蓝染色，表明大多数OK-432缀合的肿瘤疫苗是不能存活的。当用不同浓度的GA（0.2- 0.0002%）处理的肿瘤细胞免疫时，在高于0.02%GA的高浓度条件下的缀合强烈抑制B16黑色素瘤细胞的活力，使得直到180天都没有观察到肿瘤。OK-432缀合的肿瘤疫苗的表面被抗OK-432抗体的FITC发射强烈染色，而未处理的肿瘤细胞未染色。B16疫苗的三次免疫显示出对B16黑色素瘤发病率的最强抑制作用， ...更多信息 死亡率降低了艾德。仅有的Sq 1979疫苗免疫诱导抗Sq 1979特异性效应，而B16疫苗不引起交叉反应性抗肿瘤效应。(2)KLN-205舌癌模型的制备为了分析OK-432缀合的肿瘤疫苗的抗肿瘤作用的机制，我开发了实验性小鼠舌癌模型。两种鳞状细胞癌（KLN-205和Sq 1979细胞）进行了检查。在DBA/2小鼠舌癌模型中，KLN-205细胞2×10^5和5×10^5组的成瘤率为100%。另一方面，在Sq-1979细胞模型中，5×10^5细胞组的致癌率为100%，但观察到肿瘤消退。因此，选择2×10^5个细胞/ 40 μl KLN-205细胞接种作为合适的实验性舌癌模型。(3)KLN-205瘤苗的抗肿瘤效应分析为了分析KLN-205细胞免疫小鼠后的变化，我进行了组织学检查。接种KLN 205疫苗组24 h后组织切片可见肿瘤细胞周围有明显淋巴细胞浸润。另一方面，在对照组中显示散在淋巴细胞，未观察到浸润淋巴细胞。这些结果表明，免疫小鼠获得了对KLN-205细胞的免疫记忆。(4)KLN 205-疫苗引起细胞溶解活性在细胞溶解试验中，来自用KLN 205-疫苗免疫的小鼠的脾细胞显示出比来自用其它疫苗免疫的小鼠的脾细胞更强的细胞溶解反应。但GA处理的KLN-205、GA处理的OK-432和单独的OK-432表现出与对照几乎相同的特异性裂解水平。这些发现表明，两者（肿瘤细胞和OK-432）的缀合对于使用基于细胞的疫苗的癌症免疫治疗是重要的。少

项目成果

Li X, Bukawa H, Tsuyuki Y, Omura S, Fujita K: "Experimental mouse model with tongue cancer produced by KLN-205 cell line inoculation for development of tumor vaccine"Yokohama Medical Bulletin. 49. 25-33 (2002)

Li X、Bukawa H、Tsyuki Y、Omura S、Fujita K：“通过接种 KLN-205 细胞系产生的舌癌实验小鼠模型，用于开发肿瘤疫苗”横滨医学通报。

Li X, Bukawa H, Tsuyuki Y, Omura S, Fujita K: "Experimental mouse model with tongue cancer produced by KLN -205 cell line inoculation for development of tumor vaccine"Yokohama Medical Bulletin. 49. (2002)

Li X、Bukawa H、Tsyuki Y、Omura S、Fujita K：“通过接种 KLN -205 细胞系而产生的舌癌实验小鼠模型，用于开发肿瘤疫苗”横滨医学通报。

Li X, Bukawa H, Tsuyuki Y, Omura S, Fujita K: "Experimental mouse model with tongue cancer produced by KLN2O5 cell line inoculation for development of tumor vaccire"Yokohama Medical Bulletin. 49. 25-33 (2002)

Li X、Bukawa H、Tsyuki Y、Omura S、Fujita K：“通过接种 KLN2O5 细胞系以开发肿瘤疫苗而产生的舌癌实验小鼠模型”《横滨医学通报》。