Combining Virotherapy and Immunotherapy for Cancer

描述(申请人提供)：癌症治疗中最普遍的问题是经过手术、放疗和化疗的标准治疗后，恶性肿瘤细胞的重新生长和转移。到目前为止，基因治疗方法一直受到复制缺陷载体转导效率不足的影响。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显示出相当大的前景，特别是对局部晚期或复发的癌症。与传统的复制缺陷逆转录病毒载体相比，使用基于小鼠白血病病毒(MLV)的复制能力强的逆转录病毒载体进行基因转移已被证明是高效的，在乳腺癌、前列腺癌和胶质瘤模型中，在几周的时间内，即使载体上清液的初始接种量低至10E(4-5)总感染单位，对应的MOI低至0.001，也能在整个肿瘤中实现98%的转导。虽然各种其他复制的溶瘤病毒目前正在开发用于癌症治疗，但由于病毒的不受控制的传播可能带来的潜在风险，很少有人考虑使用具有复制能力的逆转录病毒(RCR)载体。事实上，基于MLV的RCR载体表现出显著的固有的肿瘤选择性，因为在所有被测试的正常组织中，敏感的PCR分析无法检测到瘤外扩散，这可能是由于MLV固有的无法感染静止细胞的原因，并且MLV还表现出其他有利的特征，包括简单的基因组和良好的生命周期，转录调控的保真度，以及自杀基因的高效非抒情传递，这些基因在达到最大的瘤内载体扩散和最佳给药时机之前一直稳定表达。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和体内肿瘤模型中都可以高效地杀死癌细胞。RCR载体介导的自杀基因治疗颅内胶质瘤的结果是，100%的存活时间为120天，而对照组为0%的存活时间为40天，并且在所有转移的异位病灶中观察到病毒的持续存在。我们现在提出，有效和稳定地将病毒新抗原赋予肿瘤细胞，将多种方法结合起来，为进一步提高这种治疗的长期疗效提供了机会，包括(1)通过后续注射针对病毒蛋白的放射性同位素结合抗体进行血清免疫治疗，以及(2)通过外周致敏表达病毒抗原的肿瘤疫苗，结合免疫刺激细胞因子的肿瘤内基因转移进行主动免疫治疗。在这项应用中，我们结合了多个合作者的专业知识，在体内同基因脑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.