Vaccine and Drug Combination Therapy for Human Cancers

联合疗法：疫苗化疗：包括顺铂和长春瑞滨在内的铂联合化疗是非小细胞肺癌的标准治疗方案。亚致死性接触某些化疗药物已被证明改变了人类肿瘤细胞的表型或生物学，使它们更容易受到细胞毒性T淋巴细胞(CTL)介导的溶解。然而，顺铂/长春瑞滨对肿瘤T细胞毒敏感性的影响及其分子机制尚未完全阐明。我们在体外检测了这种化疗对五种不同的人肺癌细胞系的生长、细胞表面表型和CTL介导的裂解的影响，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性暴露于铂对偶可调节肿瘤细胞的表型，并增加对主要组织相容性复合体限制性穿孔素/颗粒酶介导的CTL杀伤的敏感性。这些研究还表明，暴露于化疗中显著降低了转化生长因子-β/白介素8的蛋白质分泌比率。我们检测了两个肺肿瘤细胞系的基因表达谱，以确定对亚致死性顺铂/长春瑞滨反应的共同基因特征，并发现只有16个转录本协同表达，包括细胞因子/趋化因子表达和细胞凋亡，如肿瘤坏死因子-α、白介素8、CXCL5和B细胞淋巴瘤-2样基因(BCL-2)。总体而言，这些结果表明，顺铂/长春瑞滨亚致死性暴露通过调节(A)肿瘤表型、(B)细胞因子/趋化因子环境和(C)促凋亡/抗凋亡基因比率，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种与免疫原性细胞死亡截然不同并与之互补的复杂机制。这一分子标记可能有助于预测免疫治疗的反应，并为疫苗与顺铂/长春瑞滨方案联合使用的潜在临床益处提供理论基础。联合治疗：小分子抑制剂与疫苗：小分子bcl2抑制剂正在几种类型的肿瘤的I/II期临床试验中作为单一疗法进行检验。然而，关于bcl2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bcl2抑制物对免疫功能的影响，并确定该抑制物与重组疫苗联合治疗肿瘤的最有效方法。研究了PAN-bcl2抑制剂GX15-070对处于两个不同活化阶段的小鼠CD8T淋巴细胞和调节性T淋巴细胞(Treg)的体外作用。分析重组痘苗/鸡痘CEA-Tricom疫苗接种GX15-070对荷瘤小鼠的肿瘤浸润性淋巴细胞和脾细胞的体内效应。这种序贯治疗的疗效是通过减少肺部肿瘤结节来衡量的。与早期激活的细胞相比，激活的成熟CD8T细胞对GX15-070的抵抗力更强。经bcl2抑制剂治疗后，Treg功能明显下降。体内接种后给予GX15-070，以避免对疫苗免疫的诱导产生负面影响，导致瘤内活化的CD8/Treg比值升高，肺部肿瘤结节明显减少。这项研究首次显示了一种小分子bcl2抑制剂对免疫系统和疫苗的影响。这也是第一次证明该序列在小鼠模型中减少肿瘤的有效性，为联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孙尼替尼。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相结合的治疗实体瘤的平台。利用小鼠模型，我们研究了在人体内给予相当于37.5-50毫克/天的浓度的舒尼替尼4周，然后停药2周(舒尼替尼4/2)的效果。我们评估了不同时间组合的舒尼替尼和编码癌胚抗原(CEA)的痘病毒疫苗加上3个共刺激分子对CEA转基因(CEA-TG)小鼠免疫反应的影响。在CEA-TG小鼠体内进行了抗肿瘤研究，携带CEA基因的MC38小鼠结肠癌(MC38-CEA)，同时或连续用舒尼替尼和疫苗治疗。在体外，舒尼替尼抑制MC38-CEA细胞上PDGFR磷酸化的浓度与人类治疗期间的生物可用浓度相似。在体内，一个周期的舒尼替尼4/2引起双峰免疫效应：(A)在治疗4周期间调节细胞减少，(B)在治疗中断2周期间免疫抑制反弹。在CEA-TG小鼠CEA+肿瘤的模型中，持续的舒尼替尼和疫苗增加了肿瘤内抗原特异性T淋巴细胞的渗透，减少了免疫抑制T调节细胞和髓系来源的抑制细胞，减少了肿瘤体积，并提高了存活率。持续服用舒尼替尼的免疫调节活性可以创造一个更允许免疫的环境。结合免疫疗法，苏尼替尼治疗应先于疫苗治疗，以便对免疫系统进行预适应，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.