肿瘤细胞治疗研究
英文原题：Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time.
这是首次使用靶向 meso 和 MUC16 的串联 CAR 设计，并证明其在肿瘤控制上优于单特异性 CAR-T 细胞。
背景：肿瘤异质性和抗原逃逸是嵌合抗原受体（CAR）T细胞疗法的耐药机制，在实体瘤中尤其突出。在异质性肿瘤模型中，采用单一CAR构建体同时靶向多个抗原，可能比单特异性CAR-T细胞更好地控制肿瘤。为克服肿瘤异质性，我们采用串联CAR设计，同时靶向间皮素（meso）和Mucin 16（MUC16），这两种抗原常表达于实体瘤。 方法：我们基于多种抗meso（SS1）和抗MUC16胞外结构域（MUC16ecto，4H11）单链可变片段（scFv）组合及不同G4S连接肽长度，设计了一系列串联CAR构建体。随后根据其对可溶性抗原的结合、空间位阻、亲合力以及体外针对表达一种或两种抗原细胞系的功能，确定最佳串联CAR设计。最后，在体外（二维和三维模型）及体内混合肿瘤模型中，将串联CAR与单特异性CAR-T细胞进行比较。 结果：我们发现，scFv排列方式和连接肽长度会影响抗原结合及T细胞CAR表达。串联CAR构型TanCAR1（SS1 scFv位于远端，两个scFv间采用一个G4S重复作为连接肽）体外结合和活化特征最佳；在体内外混合肿瘤模型中，其表现优于SS1和4H11单特异性CAR-T细胞，并根据抗原密度呈现抗原驱动的肿瘤细胞杀伤。此外，使用不同抗原表达水平的肿瘤细胞进行声学力显微镜分析发现，TanCAR1 T细胞可能一次结合一种抗原，而非同时结合两种。 结论：这是首次采用串联CAR设计同时靶向meso和MUC16，并证明其肿瘤控制效果优于单特异性CAR-T细胞。靶向meso和MUC16ecto的串联CAR-T细胞可用于克服卵巢癌和胰腺癌的肿瘤细胞异质性；其一次结合一种抗原及依赖抗原密度的肿瘤细胞杀伤特性，也有助于设计治疗策略。
BACKGROUND: Tumor heterogeneity and antigen escape are mechanisms of resistance to chimeric antigen receptor (CAR)-T cell therapy, especially in solid tumors. Targeting multiple antigens with a unique CAR construct could be a strategy for a better tumor control than monospecific CAR-T cells on heterogeneous models. To overcome tumor heterogeneity, we targeted mesothelin (meso) and Mucin 16 (MUC16), two antigens commonly expressed in solid tumors, using a tandem CAR design. METHODS: We designed a series of tandem CAR constructs based on various anti-meso (SS1) and anti-MUC16 ectodomain (MUC16ecto) (4H11) single-chain variable fragment (scFv) arrangements and G4S linker lengths. Then we determined the best tandem CAR design based on binding of soluble antigens, steric hindrance, avidity and functionality against cell lines expressing one or both antigens in vitro. Finally, we compared the tandem CAR to monospecific CAR-T cells in mixed tumor models in vitro (two-dimensional and three-dimensional models) and in vivo. RESULTS: We show that the scFv arrangement and linker length impacted antigen binding and CAR expression in T cells. Tandem CAR configuration (TanCAR1) (with SS1 scFv located distally and one G4S repeat as the linker between scFvs) had the best binding and activation profile in vitro and outperformed SS1 and 4H11 monospecific CAR-T cells in mixed tumor models in vitro and in vivo, showing an antigen-driven killing of tumor cells based on antigen density. Moreover, acoustic force microscopy, using tumor cells with different levels of antigen expression, revealed that TanCAR1-T cells likely bind to one antigen at a time rather than simultaneously. CONCLUSIONS: This is the first time using a tandem CAR design targeting meso and MUC16, and demonstrating a benefit on tumor control over monospecific CAR-T cells. Tandem CAR-T cells targeting meso and MUC16ecto could be employed as a strategy to overcome tumor cell heterogeneity in ovarian and pancreatic tumors, and may help to design therapeutic approaches relying on its one-antigen-at-a-time binding properties and on its antigen-driven killing of tumor cells based on antigen density.
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