Combining Virotherapy and Immunotherapy for Cancer

描述（由申请人提供）：癌症治疗中最普遍的问题是手术、放疗和化疗标准治疗后恶性细胞的再生长和转移。基因治疗方法已经遭受到迄今为止使用的复制缺陷型载体的不足的转导效率。复制能力载体代表了一种新兴技术，作为一种新的治疗选择，特别是对于局部晚期或复发性癌症，显示出相当大的希望。与常规的复制缺陷型逆转录病毒载体相比，使用基于可复制小鼠白血病病毒（MLV）的逆转录病毒载体的基因转移已被证明是高效的，在乳腺癌、前列腺癌和神经胶质瘤模型中，即使初始接种的载体上清液低至10 e（4-5）个总感染单位，对应于低至0.001的MOI。虽然各种其他复制型溶瘤病毒目前正在开发作为癌症治疗剂，但由于可能与病毒不受控制的传播相关的潜在风险，很少考虑使用可复制逆转录病毒（RCR）载体。事实上，基于MLV的RCR载体表现出显著程度的固有肿瘤选择性，因为在所有测试的正常组织中通过灵敏的PCR测定法检测不到肿瘤外扩散，这可能是由于MLV内在地不能感染静止细胞，并且MLV表现出另外的有利特征，包括简单的基因组和良好表征的生命周期，转录调节的保真度，和稳定表达的自杀基因的有效非抒情传递，直到达到最大的肿瘤内载体扩散和前药给药的最佳时机。使用表达自杀基因胞嘧啶脱氨酶的RCR载体，我们现在已经证明了在培养物和体内肿瘤模型中高效杀死癌细胞。用RCR载体介导的自杀基因疗法治疗颅内胶质瘤导致100%存活>120天，而对照组的存活率为0%，存活时间< 40天，并且在所有转移性异位病灶中观察到病毒持续存在。我们现在提出，将病毒新抗原有效且稳定地赋予肿瘤细胞提供了通过组合多种方法进一步改善该疗法的长期功效的机会，所述方法包括（1）通过后续施用针对病毒蛋白的放射性同位素缀合的抗体的血清免疫疗法，和（2）通过用表达病毒抗原的肿瘤疫苗进行外周致敏的主动免疫疗法，结合免疫刺激细胞因子的肿瘤内基因转移。在本申请中，我们结合了多个合作者的专业知识，在体内同基因颅内胶质瘤模型中直接测试这些方法。

DESCRIPTION (provided by applicant): The most prevalent problem in cancer therapy is the re-growth and metastasis of malignant cells after standard treatment with surgery, radiation, and chemotherapy. Gene therapy approaches have suffered from the inadequate transduction efficiencies of replication-defective vectors that have been used thus far. Replication-competent vectors represent an emerging technology that shows considerable promise as a novel treatment option, particularly for locally advanced or recurrent cancer. In contrast to conventional replication-defective retrovirus vectors, gene transfer using replication-competent murine leukemia virus (MLV)-based retrovirus vectors has proven to be highly efficient, resulting in >98% transduction throughout entire tumors over a period of several weeks in breast cancer, prostate cancer, and glioma models, even with an initial inoculum of vector supematant as low as 10e(4-5) total infectious units, corresponding to MOIs as low as 0.001. While various other replicating oncolytic viruses are now in development as cancer therapeutics, the use of replication-competent retrovirus (RCR) vectors has rarely been contemplated due to the potential risks that might be associated with uncontrolled spread of virus. In fact, MLV-based RCR vectors exhibit a significant degree of inherent tumor-selectivity, as extratumoral spread was undetectable by sensitive PCR assays in all normal tissues tested, presumably due to the intrinsic inability of MLV to infect quiescent cells, and MLV exhibits additional advantageous characteristics including a simple genome and well-characterized life cycle, fidelity of transcriptional regulation, and efficient non-lyric transmission of suicide genes which are stably expressed until maximal intratumoral vector spread and optimal timing for pro-drug administration is reached. Using RCR vectors expressing the suicide gene cytosine deaminase, we have now demonstrated highly efficient killing of cancer cells both in culture and in tumor models in vivo. Treatment of intracranial gliomas with RCR vector-mediated suicide gene therapy resulted in 100% survival for >120 days, compared to 0% survival of control groups in < 40 days, and viral persistence was observed in all metastatic ectopic foci. We now propose that the efficient and stable conferral of viral neo-antigens to the tumor cells present an opportunity to further improve the long-term efficacy of this therapy by combining multiple approaches, including (1) seroimmunotherapy by follow-up administration of radioisotope-conjugated antibodies directed against viral proteins, and (2) active immunotherapy by peripheral sensitization with tumor vaccines expressing viral antigens, combined with intratumoral gene transfer of immunostimulatory cytokines. In this application, we have combined the expertise of multiple collaborators to directly test these approaches in syngeneic intracranial glioma models in vivo.