Vaccine and Drug Combination Therapy for Human Cancers

联合治疗：疫苗化疗：铂双联化疗，包括顺铂加长春瑞滨，是非小细胞肺癌的标准治疗。已证明亚致死暴露于某些化疗剂可改变人肿瘤细胞的表型或生物学，使其对细胞毒性T淋巴细胞（CTL）介导的裂解更敏感。然而，顺铂/长春瑞滨对肿瘤对T细胞毒性的敏感性的影响及其分子机制尚未完全阐明。我们研究了这种化疗对5种不同的人肺癌细胞系的生长、细胞表面表型和CTL介导的体外裂解的影响，并研究了与增强CTL敏感性相关的分子机制。这些研究表明，人肺肿瘤细胞亚致死暴露于铂双联体调节肿瘤细胞表型，并增加对主要组织相容性复合物限制性穿孔素/颗粒酶介导的CTL杀伤的敏感性。这些研究还表明，暴露于化疗显着降低转化生长因子-β/白细胞介素（IL）-8的蛋白质分泌比。我们检测了两种肺肿瘤细胞系的基因表达谱，以确定对亚致死顺铂/长春瑞滨的响应的共享基因签名，并发现仅16种转录物的协调表达，包括细胞因子/趋化因子表达和细胞凋亡的转录物，如肿瘤坏死因子-α、IL 8、CXCL 5和B细胞淋巴瘤-2样基因（BCL-2）。总体而言，这些结果表明，亚致死暴露于顺铂/长春瑞滨通过调节（a）肿瘤表型，（B）细胞因子/趋化因子环境和（c）促凋亡/抗凋亡基因比例增加了对穿孔素/颗粒酶介导的CTL杀伤的敏感性。这里提供的数据提出了一个复杂的机制，这是不同的和互补的免疫原性细胞死亡。这种分子特征可能是有用的，在预测免疫治疗的反应，以及提供的潜在临床效益的联合使用疫苗与顺铂/长春瑞滨regiments.Combination治疗的理由：小分子抑制剂与疫苗：小分子BCL-2抑制剂正在检查作为单一疗法在I/II期临床试验的几种类型的肿瘤。然而，关于BCL-2抑制剂对免疫功能的影响的数据很少。本研究的目的是研究小分子BCL-2抑制剂对免疫功能的影响，并确定将该抑制剂与重组疫苗结合治疗肿瘤的最有效方法。在2个不同活化阶段的小鼠CD 8 T淋巴细胞以及调节性T淋巴细胞（Treg）中评估了泛BCL-2抑制剂GX 15 -070的体外作用。在携带肺肿瘤的小鼠的肿瘤浸润淋巴细胞和脾细胞中分析重组牛痘/鸡痘CEA-TRICOM疫苗接种后GX 15 -070的体内作用。这种序贯疗法的治疗效果被测量为肺肿瘤结节的减少。与早期活化的细胞相比，活化的成熟CD 8 T淋巴细胞对GX 15 -070更具抗性。Treg功能在用BCL-2抑制剂治疗后显著降低。在体内，在疫苗接种后给予GX 15 -070，以便不会对疫苗介导的免疫的诱导产生负面影响，从而导致肿瘤内活化的CD 8：Treg比率增加，并且肺肿瘤结节显著减少。这项研究是第一个显示小分子BCL-2抑制剂对免疫系统和疫苗的影响。这也是第一个证明这种序列在小鼠模型中减少肿瘤的有效性，为组合临床研究的设计提供了理论基础。在另一组研究中，我们研究了酪氨酸激酶的小分子抑制剂舒尼替尼。本研究旨在探讨舒尼替尼的免疫调节作用，以期合理设计与免疫疗法联合治疗实体瘤的平台。使用小鼠模型，我们研究了舒尼替尼在人体中以37.5-50 mg/天的浓度给药4周，然后停药2周（舒尼替尼4/2）的效果。我们评估了舒尼替尼和编码癌胚抗原（CEA）的痘病毒疫苗加3种共刺激分子的不同时间组合对CEA转基因（CEA-Tg）小鼠免疫应答的影响。在携带CEA转染的MC 38鼠结肠癌（MC 38-CEA）的CEA-Tg小鼠中进行抗肿瘤研究，同时或顺序使用舒尼替尼和疫苗治疗。在体外，舒尼替尼抑制MC 38-CEA细胞上的PDGFR磷酸化，浓度与人体治疗期间的生物学可用浓度相似。在体内，舒尼替尼4/2的一个周期引起双峰免疫效应：（a）在4周治疗期间调节细胞减少和（B）在2周治疗中断期间免疫抑制反弹。在使用携带CEA+肿瘤的CEA-Tg小鼠的模型中，连续舒尼替尼后接种疫苗增加了抗原特异性T淋巴细胞的瘤内浸润，减少了免疫抑制性T调节细胞和髓源性抑制细胞，减少了肿瘤体积，并增加了生存期。舒尼替尼连续给药的免疫调节活性可以创造一个更免疫的环境。在与免疫疗法的组合中，舒尼替尼治疗应先于疫苗，以预处理免疫系统，以最大限度地提高对疫苗介导的免疫增强的反应。

Combination Therapy: Chemotherapy with vaccine:Chemotherapy with platinum doublets, including cisplatin plus vinorelbine, is standard of care for non-small-cell lung cancer. Sublethal exposure to certain chemotherapeutic agents has been demonstrated to alter the phenotype or biology of human tumor cells, rendering them more susceptible to cytotoxic T lymphocyte (CTL)-mediated lysis. The effects of cisplatin/vinorelbine on tumor sensitivity to T-cell cytotoxicity and its molecular mechanisms, however, have not been fully elucidated. We examined the effect of this chemotherapy on growth, cell-surface phenotype, and CTL-mediated lysis of five distinct human lung carcinoma cell lines in vitro and examined the molecular mechanisms associated with enhanced CTL sensitivity. These studies demonstrate that sublethal exposure of human lung tumor cells to the platinum doublet modulates tumor cell phenotype and increases sensitivity to major histocompatibility complex-restricted perforin/granzyme-mediated CTL killing. These studies also demonstrate that exposure to chemotherapy markedly decreased the protein secretion ratio of transforming growth factor-beta/interleukin (IL)-8. We examined the gene expression profile of two lung tumor cell lines to identify a shared gene signature in response to sublethal cisplatin/vinorelbine and found coordinate expression of only 16 transcripts, including those for cytokine/chemokine expression and apoptosis such as tumor necrosis factor-alpha, IL8, CXCL5, and B cell lymphoma-2-like genes (BCL-2). Overall, these results suggest that sublethal exposure to cisplatin/vinorelbine increases sensitivity to perforin/granzyme-mediated CTL killing by modulation of (a) tumor phenotype, (b) cytokine/chemokine milieu, and (c) the proapoptotic/antiapoptotic gene ratio. The data presented here propose a complex mechanism that is distinct from and complementary to that of immunogenic cell death. This molecular signature may be useful in predicting responses to immunotherapy as well as provide the rationale for the potential clinical benefit of the combined use of vaccine with cisplatin/vinorelbine regimens.Combination Therapy: Small-Molecule Inhibitors with vaccine:Small molecule BCL-2 inhibitors are being examined as monotherapy in phase I/II clinical trials for several types of tumors. However, few data are available about the effect of BCL-2 inhibitors on immune function. The aims of this study were to investigate the effect of a small molecule BCL-2 inhibitor on immune function and determine the most effective way of combining this inhibitor with a recombinant vaccine to treat tumors. The in vitro effect of the pan-BCL-2 inhibitor GX15-070 was assessed in mouse CD8 T lymphocytes at 2 different stages of activation as well as regulatory T lymphocytes (Treg). The in vivo effect of GX15-070 after recombinant vaccinia/fowlpox CEA-TRICOM vaccination was analyzed in tumor-infiltrating lymphocytes, and in splenocytes of mice bearing pulmonary tumors. The therapeutic efficacy of such sequential therapy was measured as a reduction of pulmonary tumor nodules. Activated mature CD8 T lymphocytes were more resistant to GX15-070 as compared to early-activated cells. Treg function was significantly decreased after treatment with the BCL-2 inhibitor. In vivo, GX15-070 was given after vaccination so as to not negatively impact the induction of vaccine-mediated immunity, resulting in increased intratumoral activated CD8:Treg ratio, and significant reduction of pulmonary tumor nodules. This study is the first to show the effect of a small molecule BCL-2 inhibitor on the immune system and following a vaccine. It is also the first to demonstrate the efficacy of this sequence in reducing tumors in mouse models, providing a rationale for the design of combinational clinical studies.In a different set of studies, we investigated the a small-molecule inhibitor for tyrosine kinase; sunitinib. This study investigated the immunomodulatory effects of sunitinib in order to rationally design combinational platforms with immunotherapies for the treatment of solid tumors. Using a mouse model, we studied the effects of sunitinib given for 4 weeks at concentrations comparable to 37.5-50 mg/day in humans, followed by 2 weeks off the drug (sunitinib 4/2). We assessed the effect of differently timed combinations of sunitinib and a poxvirus-based vaccine encoding carcinoembryonic antigen (CEA) plus 3 costimulatory molecules on immune responses in CEA-transgenic (CEA-Tg) mice. Antitumor studies were performed in CEA-Tg mice bearing CEA-transfected MC38 murine colon carcinomas (MC38-CEA), treated either concurrently or sequentially with sunitinib and vaccine. In vitro, sunitinib inhibited PDGFR phosphorylation on MC38-CEA cells at concentrations similar to those biologically available during human treatment. In vivo, one cycle of sunitinib 4/2 caused bimodal immune effects: (a) decreased regulatory cells during the 4 weeks of treatment and (b) an immune-suppression rebound during the 2 weeks of treatment interruption. In a model using CEA-Tg mice bearing CEA+ tumors, continuous sunitinib followed by vaccine increased intratumoral infiltration of antigen-specific T lymphocytes, decreased immunosuppressant T regulatory cells and myeloid-derived suppressor cells, reduced tumor volumes, and increased survival. The immunomodulatory activity of continuous sunitinib administration can create a more immune-permissive environment. In combination with immunotherapies, sunitinib treatment should precede vaccine, in order to precondition the immune system, to maximize the response to vaccine-mediated immune enhancement.