Improved idiotype immunotherapy for lymphoma by RNA vaccine delivery

描述(申请人提供)：近年来，非霍奇金淋巴瘤(NHL)已上升为美国最常见的血液系统恶性肿瘤。积极的新联合化疗方案缓慢提高了大多数疾病阶段的缓解率，但即使患者在治疗过程中获得缓解，复发的风险仍然很大，而且复发的患者的特点是对治疗的反应降低。为了改善患者的生存结果，人们一直关注通过接种一种肿瘤特异性抗原：细胞表面表达的免疫球蛋白(Ig)，或独特型来产生积极的、肿瘤特异性的免疫反应。临床研究表明，当患者对疫苗产生免疫反应时，他们的生存特征有所改善。然而，蛋白质Ig很难针对患者的特定应用而制造，并且可能不能为荷瘤患者提供足够的免疫刺激。我们的假设是，基于RNA的疫苗可以通过在病毒抗原递送的背景下传递独特型序列来提高抗独特型免疫，这种系统非常适合简单快速的个性化疫苗生产。我们的目标是设计和测试表达抗原的RNA模型构建物，以确定基于RNA的独特型疫苗在两种非霍奇金淋巴瘤小鼠模型(38C13和A20)中的有效性。这些模型是理想的，因为它们需要抗体(38C13)或CTL激活(A20)来实现最佳的肿瘤保护，这些特征也是成功的患者免疫激活所必需的。我们将使用塞姆利基森林病毒(SFV)非结构基因来驱动38C13和A20独特型序列的表达，无论是单独还是与细胞因子基因序列一起表达，以实现最大限度的抗原传递和免疫激活。将利用烟草花叶病毒外壳蛋白的新的自组装特性对SFV RNA进行封装，而不是在细胞培养中使用天然衣壳进行封装。制备囊化的RNA疫苗很简单，只需将外壳蛋白与RNA混合就能产生完全保护的假病毒颗粒。包装的RNA伪病毒疫苗诱导抗体和细胞毒性T淋巴细胞(CTL)激活，以及肿瘤保护性免疫，而不需要额外的佐剂。疫苗制剂可以被表面多肽“包裹”，降低对病毒外壳蛋白的免疫反应，并稳定地重复传递疫苗抗原和加强免疫。我们将建立最好的共传递细胞因子基因的方法，以促进提高免疫激活和逆转对38C13和A20 NHL肿瘤抗原的自身抗原耐受。将在体外验证抗原表达水平和细胞因子活性，以及在体内验证抗体和CTL对靶抗原的反应性。我们将测试可能增强抗独特型免疫或提高疫苗效力的多肽抗原结合物。最后，我们将在体内测试最有效的独特型抗原/细胞因子包裹的RNA配方，并在相关的38C13或A20小鼠肿瘤模型中将提高的免疫力与肿瘤保护相关联。公共卫生相关性：通过合理的药物设计来改进癌症疫苗治疗是研究科学家和医学界的高度优先事项。这个R21应用程序寻求通过利用对RNA病毒的先天免疫来实现改进的癌症疫苗特性。我们的假设是，表达非霍奇金淋巴瘤肿瘤抗原的封装RNA疫苗将提供优越的免疫激活和对淋巴瘤肿瘤生长的保护。与目前的疗法不同，定制疫苗很容易制造，可能会促进个性化患者免疫疗法的广泛采用。

DESCRIPTION (provided by applicant): In recent years, Non-Hodgkin's lymphoma (NHL) has risen to be the most common hematologic malignancy in the United States. Aggressive new combination chemotherapy regimes have slowly improved remission rates for most stages of disease, however the risk of relapse is still significant, even if patients achieve remission during therapy, and patients that relapse are characterized by a reduced response to treatment. To improve patient survival outcomes, significant attention has been focused on the generation of an active, tumor specific, immune response through vaccination with a tumor-specific antigen: the cell-surface expressed immunoglobulin (Ig), or idiotype. Clinical studies show that when patients make an immune response to the vaccine, they have improved survival characteristics. Protein Ig, however, is difficult to make for patient specific applications, and may not provide adequate immune stimulation in tumor-bearing patients. Our hypothesis is that RNA based vaccines can improve anti-idiotype immunity by delivering idiotype sequences in the context of viral antigen delivery, in a system that is ideally suited for simple and rapid individualized vaccine production. Our goal is to design and test model antigen expressing RNA constructs to determine the efficacy of RNA based idiotype vaccines in two murine models of NHL (38C13 and A20). These models are ideal, because they require either antibody (38C13) or CTL activation (A20) for optimal tumor protection, characteristics that are also needed for successful patient immune activation. We will use Semliki Forest Virus (SFV) nonstructural genes to drive expression of 38C13 and A20 idiotype sequences, either singly or in tandem with cytokine gene sequences for maximum antigen delivery and immune activation. SFV RNA, rather than encapsidated in cell culture with native capsid, will be encapsidated using the novel self-assembly properties of Tobacco Mosaic Virus coat protein. Preparation of encapsidated RNA vaccines is simple, involving merely mixing coat protein with RNA to generate fully protected pseudovirus particles. Encapsidated RNA pseudovirus vaccines induce both antibody and cytotoxic T lymphocyte (CTL) activation, as well as tumor protective immunity, without additional adjuvants. Vaccine agents can be "cloaked" with surface peptides reduce immune responses to viral coat protein, and are stable for repeated vaccine antigen delivery and boosting. We will establish the best method to co-deliver cytokine genes to promote improved immune activation and reversal of self-antigen tolerance to 38C13 and A20 NHL tumor antigens. Antigen expression level and cytokine activity will be verified in vitro, as well as antibody and CTL reactivity to target antigens in vivo. We will test peptide antigen conjugates that may boost anti-idiotype immunity or improve vaccine potency. Lastly, we will test the most effective idiotype antigen/cytokine encapsidated RNA formulations in vivo and correlate improved immunity with tumor protection in the relevant 38C13 or A20 mouse tumor model. PUBLIC HEALTH RELEVANCE: Improving cancer vaccine therapy by rational drug design is a high priority of research scientists and the medical community. This R21 application seeks to implement improved cancer vaccine characteristics by harnessing innate immunity to RNA viruses. Our hypothesis is that encapsidated RNA vaccines that express non-Hodgkin's lymphoma tumor antigens will provide superior immune activation and protection against lymphoma tumor growth. Unlike current therapies, custom vaccines are easily made, and may facilitate widespread adoption of individualized patient immunotherapy.