Expressing New Tumor Antigens by Inhibition of Nonsense Mediated mRNA Decay

描述（由申请人提供）：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，肿瘤不表达能被免疫系统识别为“外源”的强效肿瘤抗原。目前开发基于免疫的模式的重点是增强针对肿瘤中表达的现有（尽管较弱）抗原的免疫反应。另一种方法——这一建议的重点——是在肿瘤细胞中原位表达新的、因此有效的抗原。阻碍这种策略发展的两个主要挑战，如何在癌症患者的播散性肿瘤病变中表达新的抗原，以及如何限制这些抗原在肿瘤中的表达，从而阻止它们在正常组织中的表达，通过本提案所描述的方法得到解决。新抗原在肿瘤细胞中的表达将使用siRNA技术来抑制无义介导的mRNA衰变（NMD），这是一种阻止含有过早终止密码子的mRNA表达的监视机制。针对肿瘤细胞的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体配体组成的新型靶向技术实现。与抗体不同，适体或适体靶向siRNA偶联物可以在化学过程中合成，为生产临床级试剂提供了更直接和更具成本效益的制造和监管审批过程。本建议所检验的假设（得到广泛初步研究的支持）是：(1)。肿瘤细胞中的NMD抑制将导致新产物的表达，这些新产物将作为肿瘤排斥抗原发挥作用，引发免疫反应，对肿瘤生长产生负面影响，以及（II）。在荷瘤小鼠中，适配体靶向siRNA对NMD的抑制足以抑制肿瘤生长。在临床前小鼠模型中进行的拟议研究的主要目的是提供概念证明，使用适配体- sirna偶联物对NMD的肿瘤靶向抑制将引发肿瘤免疫，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。本建议的具体目的是：(1)。开发并表征体外最佳的PSMA适体靶向sirna，这些sirna与小鼠nmd特异性因子相对应。（2）. 评价PSMA适体- sirna偶联物对荷瘤小鼠抗肿瘤免疫的作用。(3)。开发Her2适配体- sirna偶联物，并评估其在可移植和自发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依据和指导。

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.