Chaperone rich cell lysates (CRCL)Natural adjuvants and*

描述（由申请人提供）：尽管癌症化疗的剂量不断增加，越来越多的根治性手术，卵巢癌的总死亡率仍然没有改变，也是不可接受的。因此，明确需要新的生物学方法来治疗这种毁灭性的肿瘤。我们开发了一种新的方法，利用自由溶液等电聚焦，有效地从肿瘤裂解物中富集多种伴侣复合物。我们已经证明，富含伴侣蛋白的细胞裂解液（CRCL）疫苗接种比纯化个体伴侣蛋白（热休克蛋白，HSPs）免疫更有效。通过将CRCL装载到树突状细胞（dc）上，可以进一步增强CRCL的抗原性，从而即使在已有疾病的情况下也能对小鼠肿瘤产生保护作用。与暴露于未分离的肿瘤裂解液或纯化的热休克蛋白的dc相比，在小鼠肿瘤源性CRCL存在下的dc成熟并发展出优越的免疫刺激能力。我们假设这种增强的免疫原性的本质可能在于抗原肽范围的扩大，这些抗原肽通过CRCL护送到dc，并由CRCL直接激活dc。人类dc在对CRCL的反应中所经历的表型和功能变化的全部范围仍有待观察和研究。我们进一步假设，CRCL脉冲dc是有效的免疫刺激剂，可产生肿瘤特异性细胞毒性T淋巴细胞（CTL）对抗卵巢癌。CRCL脉冲DCs是一种很有前途的抗癌疫苗，可能被证明是卵巢癌妇女的有用辅助治疗。为了更好地了解CRCL- dcs作为卵巢癌潜在的天然免疫增强方法的作用，我们提出了以下具体目标：1)生成并生物化学表征卵巢癌衍生的CRCL。2)评估卵巢癌衍生的CRCL对DC的影响，以确定CRCL是否诱导成熟和/或改变DC功能3)检查卵巢癌衍生的CRCL脉冲DC产生肿瘤特异性ctl的潜力。我们将从卵巢癌中分离CRCL和纯化热休克蛋白，通过SDS-PAGE和Western blotting对其进行特异性蛋白含量分析，并评估这些疫苗的产量、稳定性和内毒素含量。卵巢癌CRCL和纯化热休克蛋白对血液和腹水来源的dc的影响将被评估。将研究DC免疫表型、细胞因子产生、吞噬活性和免疫刺激功能的变化。在腹水中发现的免疫抑制细胞因子的作用将被解决。我们将研究卵巢癌衍生的CRCL和纯热休克蛋白作为抗原来源的效力，自体（与异体）CRCL的重要性，血液衍生的DC与腹水衍生的DC在刺激CTL方面的功能，以及在CRCL脉冲DC刺激后腹水中发现的细胞因子对CTL生成的抑制作用。

DESCRIPTION (provided by applicant): Despite dose escalation of cancer chemotherapy and increasingly radical surgery, the overall mortality from ovarian cancer remains unaltered and unacceptable. Therefore there is a definite need for new biologic approaches against this devastating tumor. We have developed a novel method that efficiently enriches for multiple chaperone complexes from tumor lysates using free solution isoelectric focusing. We have documented that chaperone rich cell lysate (CRCL) vaccination is more effective than immunization with purified individual chaperones (heat shock proteins, HSPs). The antigenicity of CRCL can be augmented further by loading them onto dendritic cells (DCs) resulting in protection against murine tumors even in the setting of pre-existing disease. DCs, in the presence of murine tumorderived CRCL, mature and develop superior immunostimulatory capacity when compared to DCs exposed to unfractionated tumor lysate or purified HSPs. We hypothesize that the nature of this enhanced immunogenicity may lie in a broadened range of antigenic peptides that are escorted via the CRCL to DCs, and to the direct activation of DCs by the CRCL. The full range of phenotypic and functional changes that human DCs undergo in response to CRCL remains to be seen and will be studied. We further hypothesize that CRCL pulsed DCs are potent immunostimulants effective in generating tumor specific cytotoxic T lymphocytes (CTL) against ovarian cancer. CRCL pulsed DCs is a promising anti-cancer vaccine that may prove to be useful adjuvant therapy for women suffering from ovarian cancer. To better understand the role of CRCL-DCs as a potential natural immunoaugmentative approach for ovarian cancer, we propose the following specific aims: 1) Generate and biochemically characterize ovarian cancer-derived CRCL. 2) Evaluate the effects of ovarian cancer-derived CRCL on DCs to determine if CRCL induces maturation and/or alters DC function 3) Examine the potential of ovarian cancer-derived CRCL-pulsed DCs to generate tumor specific CTLs. We will isolate CRCL and purified HSPs from ovarian cancers, analyze them by SDS-PAGE and Western blotting for specific chaperone protein content, and assess the yield, stability and endotoxin content of these vaccines. The effects of ovarian cancer CRCL and purified HSPs on blood and ascites derived DCs will then be evaluated. Changes in DC immunophenotype, cytokine production, endocytic/phagocytic activity and immunostimulatory function will be studied. The role of immunosuppressive cytokines found in ascites will be addressed. We will study the potency of ovarian cancer derived CRCL and pure HSPs as antigen sources, the importance of autologous (versus allogeneic) CRCL, the function of blood-derived versus ascites-derived DC in stimulating CTL and the inhibitory effects of cytokines found in i ascites on CTL generation following stimulation by CRCL pulsed DCs.