Expressing New Tumor Antigens by Inhibition of Nonsense Mediated mRNA Decay

描述(申请人提供)：肿瘤不受癌症患者免疫系统控制的主要原因是，与病原体不同，肿瘤不表达可被免疫系统识别为“外来”的有效肿瘤抗原。目前开发基于免疫的方法的重点是加强对肿瘤中表达的现有抗原的免疫反应，尽管表达的抗原很弱。另一种方法--这项提议的重点--是在原位肿瘤细胞中表达新的、因此有效的抗原。这项建议中描述的方法解决了阻碍这种策略发展的两个主要挑战，即如何在癌症患者的播散性肿瘤病变中表达新抗原，以及如何将这种抗原的表达限制在肿瘤中，排除它们在正常组织中的表达。在肿瘤细胞中表达新的抗原将使用siRNA技术来抑制无义介导的mRNA衰退(NMD)，NMD是一种监测机制，可以防止含有提前终止密码子的mRNAs的表达。靶向肿瘤细胞的siRNA抑制--由于NMD过程的组成性质和生理作用是必不可少的--将使用一种由寡核苷酸适配子配体组成的新型靶向技术来实现。与抗体不同，适体或适体靶向siRNA结合物可以通过化学过程合成，提供了更直接和更具成本效益的生产和监管批准过程，以产生临床级别的试剂。经广泛的初步研究支持，这项提案中检验的假设是：(I)。抑制肿瘤细胞中的NMD将导致新的产物的表达，这些产物将作为肿瘤排斥抗原而引发免疫反应，从而对肿瘤的生长产生负面影响，以及(Ii)。适体靶向siRNA抑制荷瘤小鼠的NMD足以抑制肿瘤生长。这项拟议的研究-在临床前小鼠模型中进行-的主要目标是提供概念证明，即使用适体-siRNA偶联物的肿瘤靶向抑制NMD将诱导肿瘤免疫，并确定与同类最佳的“传统”肿瘤疫苗接种方案相比，这种新方法在预防和逆转小鼠肿瘤进展方面的有效性如何。这项建议的具体目的是：(1)。开发和鉴定体外最佳的PSMA适配子靶向小鼠NMD特异性因子的siRNAs。(2)。目的：评价PSMA适体-siRNA结合物在荷瘤小鼠体内产生抗肿瘤免疫的能力。(3)。开发Her2适体-siRNA偶联物，并评价其在可移植和自发的小鼠乳腺癌模型中抑制肿瘤生长的能力。本申请中提出的研究的成功完成将为探索适体靶向NMD抑制以增强癌症患者的肿瘤免疫原性提供理论基础和指导方针。

DESCRIPTION (provided by applicant): The main reason why tumors are not controlled by the immune system of the cancer patient is that, unlike pathogens, tumors do not express potent tumor antigens that can be recognized by the immune system as "foreign". The current focus in developing immune-based modalities is to potentiate an immune response against the existing, albeit weak, antigens expressed in the tumor. An alternative approach - the focus of this proposal - is to express new, and hence potent, antigens in tumor cells in situ. Two main challenges that have precluded the development of such strategies, how to express new antigens in the disseminated tumor lesions of the cancer patients and how to restrict expression of such antigens to the tumor, precluding their expression in normal tissue, are addressed by the approach described in this proposal. Expression of novel antigens in tumor cells will be achieved using siRNA technology to inhibit nonsense mediated mRNA decay (NMD), a surveillance mechanism which prevents the expression of mRNAs containing a premature termination codon. Targeting siRNA inhibition to tumor cells - an essential requisite because of the constitutive nature and physiological roles of the NMD process - will be achieved using a novel targeting technology comprised of oligonucleotide aptamer ligands. Aptamers or aptamer targeted siRNA conjugates, unlike antibodies, can be synthesized in a chemical process, providing a more straightforward and cost effective manufacturing and regulatory approval process to generate clinical grade reagents. The hypotheses tested in this proposal - supported by extensive preliminary studies - are: (I). NMD inhibition in tumor cells will result in the expression of novel products which will function as tumor rejection antigens eliciting an immune response that will negatively impact on tumor growth, and (II). Aptamer-targeted siRNA inhibition of NMD in tumor-bearing mice is sufficiently robust to inhibit tumor growth. The main objectives of the proposed studies-carried out in preclinical murine models - are to provide proof-of-concept that tumor targeted inhibition of NMD using aptamer-siRNA conjugates will elicit tumor immunity, and to determine how effective is the this novel approach, compared to best-in-class "conventional" tumor vaccination protocols in preventing and reversing tumor progression in mice. The specific aims of this proposal are: (1). To develop and characterize in vitro best-in-class PSMA aptamer targeted siRNAs corresponding to murine NMD-specific factors. (2). To evaluate the ability of PSMA aptamer-siRNA conjugates to engender antitumor immunity in tumor bearing mice. (3). To develop Her2 aptamer-siRNA conjugates and evaluate their ability to inhibit tumor growth in transplantable and spontaneous murine models for breast cancer. Successful completion of the studies proposed in this application will provide the rationale and guidelines for exploring aptamer targeted NMD inhibition to potentiate tumor immunogenicity in cancer patients.