CpG ODN-MF59辅佐肿瘤-睾丸抗原诱导抗小鼠前列腺癌的作用及其机制研究

Recently, Cancer-testis antigens are thought to be good candidate for target antigen in tumor vaccine, they have been used in tumor vaccine studies widely. However, there are seldom researches focused on how to screen appropriate CTAs. In our former study, we discovered that NY-ESO-1 may be recognized by TLR4/CRT complex in the surface of immature DC to stimulated DC to produce strong anti-tumor immune responses. In a CTA screening study with large sample of prostate cancer patients, we first discovered that 4 CTAs which were NY-ESO-1, LAGE-1, SSX-2 and XAGE-1 are highly expressed in the serum of prostate cancer patients. Our project will set up prostate cancer mouse model that expressed above CTAs to study the mechanism that how these CTAs are involved in the anti-tumor immune response, and to select the CTA with strongest immunogenicity to be the target antigen in tumor vaccine. We also postulated that pathogen-associated molecular patterns or their mimics together with vehicles can increase CTA's anti-tumor capability. Therefore we choose MF59 and CpG ODN as adjuvant for CTA to study the mechanism that how they are involved in the anti-tumor immune response together. It may give new evidence in how to choose appropriate tumor vaccine adjuvant and provide new target point in the research of prostate cancer vaccine.

近年来，肿瘤-睾丸抗原（CTA）被认为是理想的肿瘤疫苗靶抗原而广泛用于肿瘤疫苗的研究，然而如何筛选合适的CTA却未见报道。我们前期研究发现：①NY-ESO-1可能与未成熟DC表面的TLR4/CRT复合体结合激活DC引起强烈的抗肿瘤免疫反应；②首次以大样本前列腺癌人群进行CTA筛查，结果显示NY-ESO-1、LAGE-1、SSX-2和XAGE-1在人前列腺血清高表达。本课题拟通过建立表达以上各种CTA的前列腺癌小鼠模型，研究它们参与肿瘤免疫应答的作用机制，选择其中免疫原性最强的CTA为疫苗靶抗原。同时提出假想：PAMPs或其模拟物与载体类佐剂联合运用能增强CTA的抗肿瘤效能。我们拟选用MF59和CpG ODN联合辅佐CTA，研究三者如何共同参与机体的抗肿瘤免疫应答，并探讨其机制。本课题的实施可望为肿瘤疫苗佐剂的选择提供新思路，为前列腺癌疫苗的研制提供新靶点。

在治疗非激素依赖性前列腺癌的动物模型和临床实践中，以树突状细胞（DC）为基础的疫苗已经呈现出满意的效果。但此类疫苗需要在体外进行繁琐的DC 培养，且耗费昂贵。因此，我们希望找到一种能够绕过体外培养DC，以体内未成熟DC为靶向的更强有力的前列腺肿瘤疫苗。首先我们要研究能诱导DC 吞噬作用和DC 在体外成熟的DC 靶向肿瘤疫苗。这种疫苗以两种形式起作用，一种是重组ESO-PSMA 融合蛋白，另一种是被辐射的编码细胞膜表面NY-ESO-1 的Myc-Cap 肿瘤细胞（叫作Myc-Cap/ESO）。然后在FVB 小鼠的移植Myc-CaP 前列腺癌模型中，评估DC 疫苗在体内抵抗CD4+/CD25+/Foxp3+调节性T 细胞（Treg）和骨髓来源的抑制性细胞（MDSC）的作用以及其抗肿瘤效能。最后阐明疫苗诱导的免疫效应和抗肿瘤效能之间的关系。.在研究中，我们成功构建了pRV-Display-ESO/ESOcs1/ESOcs2/ESOcs3/LAGE1/ HMGB1/GFP真核质粒，并可用于后续转染。成功的获得带目的基因的逆转录病毒，并将逆转录病毒转染进Myc-CaP细胞。带目的基因的Myc-CaP细胞可用于后继动物模型的建立。成功建立了Myc-CaP前列腺癌小鼠模型。接种Myc-CaP/ESO细胞的小鼠肿瘤潜伏期最短、肿瘤生长速度最快，接种Myc-CaP/ESOcs3、Myc-CaP/HMGB和Myc-CaP细胞的小鼠肿瘤潜伏期最长、肿瘤生长速度最慢。不同组小鼠肿瘤生长速度不同与针对NY-ESO-1的IgG1和IgG2a水平变化无明显关系，即与细胞免疫和体液免疫的种类无关；不同组小鼠肿瘤生长速度不同也不是由激活了不同数量的DC与MDSC所引起的。肿瘤细胞自身表达mTLR4，NY-ESO-1可能为TLRs的配体，激活TLRs并增加IL-6的分泌，促进肿瘤的生长。