Potentiating the Effects of Targeted and Cytotoxic Agents on Cell-Based Immunoth

肿瘤微环境通过限制局部肿瘤特异性T淋巴细胞的扩张，阻止T细胞的渗透和肿瘤特异性靶点的杀伤，为肿瘤免疫治疗创造了强大的障碍。因此，靶向治疗肿瘤微环境结合免疫治疗是一种合理而有吸引力的方法。我们已经证明，针对突变的BRAF的化疗和治疗都通过显著增加激活的细胞毒性T细胞(CTL)释放的颗粒酶B(GrzB)的穿孔素非依赖性通透性来增加黑色素瘤细胞对T细胞细胞毒作用的敏感性。我们的数据有力地表明，这种作用是通过在自噬过程中上调肿瘤细胞表面甘露糖-6-磷酸受体(MPR)的表达来实现的。当与化疗或靶向治疗相结合时，针对特定抗原而产生的CTL能够诱导不表达这些抗原的邻近肿瘤细胞凋亡，从而产生旁观者效应。我们的初步实验表明，具有强大的抗黑色素瘤活性的BRAF抑制剂可以诱导BFAF突变的黑色素瘤细胞MPR表达上调，这表明它们可能在过继细胞治疗中提供有效的旁观者效应。在目前的方案中，我们将进一步探索靶向和化疗药物改变肿瘤对T细胞破坏的敏感性的机制，并提出两项临床试验。在第一个试验中，我们将在治疗前后对接受靶向BRAF制剂或化疗药物的患者的可及肿瘤进行活检，并确定黑色素瘤细胞上的MPR、自噬和GrzB标记是否增加，以及在治疗开始后这些标记的增加是否可以测量。这些发现随后将转化为BRAF抑制剂维莫拉非尼联合肿瘤浸润性淋巴细胞(TIL)和淋巴枯竭后的IL-2过继细胞治疗的II期试验。这项试验将测试靶向治疗联合TIL是否会增加免疫疗法的临床疗效，并导致在接受TIL之前没有退出的患者比例更高。

The tumor microenvironment creates powerful obstacles for cancer immunotherapy by limiting the expansion of local tumor-specific T lymphocytes, preventing T-cell penetration and the killing of tumor-specific targets. Therefore, therapeutic targeting of the tumor microenvironment combined with immunotherapy is a logical and attractive approach. We have shown that both chemotherapy and treatment targeted to mutated BRAF increased the susceptibility of melanoma cells to the cytotoxic effect of T cells through a dramatic perforin-independent increase in permeability to granzyme B (GrzB) released by activated cytotoxic T cells (CTL). Our data strongly suggested that this effect was mediated via up-regulation of the expression of mannose-6-phosphate receptors (MPR) on the surface of tumor cells while undergoing autophagy. When combined with chemo- or targeted therapy, CTLs raised against specific antigens were able to induce apoptosis in neighboring tumor cells that did not express those antigens, creating a bystander effect. Our preliminary experiments demonstrated that BRAF inhibitors with potent anti-tumor activity against melanoma induced up-regulation of MPR in BFAF mutated melanoma cells, suggesting that they might provide a potent bystander effect with adoptive cell therapy. In the current proposal we will further explore the mechanisms by which targeted and chemotherapeutic agents alter tumor susceptibility to T cell destruction, and propose two clinical trials. In the first trial we will biopsy accessible tumors from patients receiving either a targeted BRAF agent or a chemotherapeutic drug before and after treatment and determine if MPR, markers of autophagy and GrzB are increased on melanoma cells, and when after initiation of treatment increases in these markers can be measured. These findings will then be translated to a phase II trial of the BRAF inhibitor vemurafenib combined with adoptive cell therapy with tumor infiltrating lymphocytes (TIL) and IL-2 after lymphdepletion. This trial will test whether targeted therapy combined with TIL will increase the clinical efficacy of the immunotherapy and result in a higher proportion of patients who do not drop out before they receive their TIL.