T cell plasticity, fusion proteins and CAR T cell-based immunotherapy of head and neck cancer

摘要 头颈部鳞状细胞癌(SCCHN)的现有治疗方法疗效有限 这促使我们设计了一种新的、有效的组合免疫疗法来治疗这种疾病。在这一策略中，T细胞 使用肿瘤抗原(TA)特异性嵌合抗原受体(CAR)作为效应器 机制，因为这种方法允许由于T淋巴细胞的肿瘤识别和自我放大的特异性 自我更新能力。我们选择了硫酸软骨素蛋白多糖4(CSPG4)作为靶点，因为I) CSPG4在约60%的SCCHN肿瘤中高表达，每个肿瘤内的异质性有限；ii) CSPG4在分化的SCCHN细胞和具有高醛脱氢酶的SCCHN细胞上均有表达 活动。后者被称为ALDHbright细胞，表现出癌症起始细胞(CICs)的特征， 因为它们是抗药性的，表达干细胞基因，并在免疫缺陷小鼠中产生肿瘤。因此， CSPG4 CAR T细胞靶向分化的SCCHN细胞和SCCHN CICs。根据癌症主干 细胞理论认为，CICs必须完全消除才能治疗成功，因为这些细胞发挥着 在疾病复发和转移扩散中起重要作用；以及iii)在正常组织中未检测到CSPG4 除了肿瘤微环境中被激活的周细胞外。因此，CSPG4与CAR的免疫靶向性 T细胞有望抑制肿瘤微环境中的新生血管生成，并有助于消除 在SCCHN细胞中，即使CSPG4低表达或缺乏表达也是如此。 在最近的研究中，我们发现CSPG4 CAR T细胞能够有效地清除CSPG4+SCCHN细胞。 在常氧条件下体外培养。此外，它们还显著抑制人SCCHN肿瘤的生长。 免疫缺陷的小鼠，但他们不能根除它们。这一提议将检验我们的工作假设，即 低氧驱动的SCCHN肿瘤恶性微环境导致CAR T细胞功能障碍并降低SCCHN 细胞对CAR T细胞介导的裂解的敏感性。T细胞的可塑性使我们i)通过以下方式恢复CAR T细胞功能 干扰PD-1/PD-L1轴以对抗CAR T细胞的耗竭和II)增强活性和抗肿瘤 选择性提高肿瘤微环境中IL-15水平对CAR T细胞活性的影响 将IL-15与抗B7-H3单抗(MAb)HEK5连接而产生的融合蛋白。此外， 通过调节抗凋亡分子恢复SCCHN细胞对CAR T细胞介导的裂解敏感性 小分子Sonidegib抑制低氧诱导的Sonic的表达水平 刺猬同源基因路径。这些实验将在体外利用SCCHN细胞系和在 免疫缺陷小鼠原位移植SCCHN细胞系和PDX(其中一些已经 已建立)。由此产生的信息将决定我们是否拥有组合策略 已开发的有效对抗SCCHN细胞利用的与缺氧相关的逃逸机制，以避免 CSPG4 CAR T细胞的识别和破坏。

ABSTRACT The limited efficacy of the available therapy for squamous cell carcinoma of the head and neck (SCCHN) has prompted us to design a novel effective combinatorial immunotherapy for this disease. In this strategy, T cells engineered with a tumor antigen (TA)-specific chimeric antigen receptor (CAR) are used as the effector mechanism, since this approach allows specificity of tumor recognition and self amplification due to T lymphocyte self renewal capacity. We have selected chondroitin sulfate proteoglycan 4 (CSPG4) as the target, since i) CSPG4 is highly expressed in about 60% of SCCHN tumors with limited heterogeneity within each tumor; ii) CSPG4 is expressed on both differentiated SCCHN cells and SCCHN cells with high aldehyde dehydrogenase activity. The latter cells referred to as ALDHbright cells, display the characteristics of cancer initiating cells (CICs), since they are drug-resistant, express stemness genes and are tumorigenic in immunodeficient mice. Therefore, CSPG4 CAR T cells target both differentiated SCCHN cells and SCCHN CICs. According to the cancer stem cell theory, CICs have to be completely eliminated for a therapy to be successful, since these cells play an important role in disease recurrence and in metastatic spread; and iii) CSPG4 is not detected in normal tissues except for activated pericytes in the tumor microenvironment. Therefore, immunotargeting of CSPG4 with CAR T cells is expected to inhibit neo-angiogenesis in the tumor microenvironment and to contribute to the elimination of SCCHN cells, even those with low or lack of CSPG4 expression. In recent studies, we have shown that CSPG4 CAR T cells are effective in eliminating CSPG4+ SCCHN cells in vitro under normoxic conditions. In addition, they significantly inhibit the growth of human SCCHN tumors in immunodeficient mice but they do not eradicate them. This proposal will test our working hypothesis that the hypoxia driven hostile microenvironment of SCCHN tumors causes CAR T cell dysfunction and reduces SCCHN cell susceptibility to CAR T cell mediated lysis. T cell plasticity allows us i) to restore CAR T cell function by disrupting PD-1/PD-L1 axis to counteract CAR T cell “exhaustion” and ii) to enhance viability and anti-tumor activity of CAR T cells by selectively increasing IL-15 level in the tumor microenvironment through the use of fusion proteins generated by linking IL-15 to anti-B7-H3 monoclonal antibody (mAb) HEK5. In addition, the susceptibility to CAR T cell mediated lysis of SCCHN cells will be restored by modulating anti-apoptotic molecule expression level through the inhibition with the small molecule sonidegib of hypoxia induced activation of Sonic Hedgehog Homolog pathway. The experiments will be performed in vitro utilizing SCCHN cell lines and in immunodeficient mice orthotopically grafted with both SCCHN cell lines and PDXs (some of which have already been established). The resulting information will determine whether the combinatorial strategy we have developed is effective in counteracting the hypoxia-related escape mechanisms utilized by SCCHN cells to avoid recognition and destruction by CSPG4 CAR T cells.