Salas-Benito Diego, Birocchi Filippo, Park Sangwoo, Ho Cassidy E, Armstrong Alexander, Parker Aiyana L, Bouffard Amanda A, Frank Jessica A, Kim Eugene, Kienka Tamina, Graham Kiana, Kelly Christopher, Goncalves Sadie, Leick Mark B, Escobar Giulia, Rueda Bo, Berger Trisha R, Maus Marcela V
Cellular Immunotherapy Program, Massachusetts General Hospital, Boston, Massachusetts, USA.
Krantz Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.
J Immunother Cancer. 2025 Sep 9;13(9):e012822. doi: 10.1136/jitc-2025-012822.
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.
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.
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.
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.
肿瘤异质性和抗原逃逸是嵌合抗原受体（CAR）-T细胞疗法耐药的机制，在实体瘤中尤为如此。在异质性模型上，使用独特的CAR构建体靶向多种抗原可能是一种比单特异性CAR-T细胞更好地控制肿瘤的策略。为了克服肿瘤异质性，我们采用串联CAR设计靶向间皮素（meso）和粘蛋白16（MUC16），这两种抗原在实体瘤中普遍表达。
我们基于各种抗间皮素（SS1）和抗MUC16胞外域（MUC16ecto）（4H11）单链可变片段（scFv）排列以及G4S接头长度设计了一系列串联CAR构建体。然后，我们根据可溶性抗原的结合、空间位阻、亲和力以及对体外表达一种或两种抗原的细胞系的功能，确定了最佳的串联CAR设计。最后，我们在体外（二维和三维模型）和体内的混合肿瘤模型中，将串联CAR与单特异性CAR-T细胞进行了比较。
我们表明，scFv排列和接头长度影响T细胞中的抗原结合和CAR表达。串联CAR构型（TanCAR1）（远端为SS1 scFv，scFv之间有一个G4S重复作为接头）在体外具有最佳的结合和激活谱，并且在体外和体内的混合肿瘤模型中优于SS1和4H11单特异性CAR-T细胞，显示出基于抗原密度的抗原驱动的肿瘤细胞杀伤作用。此外，使用具有不同抗原表达水平的肿瘤细胞的声学力显微镜显示，TanCAR1-T细胞可能一次只结合一种抗原，而不是同时结合。
这是首次使用靶向meso和MUC16的串联CAR设计，并证明在肿瘤控制方面优于单特异性CAR-T细胞。靶向meso和MUC16ecto的串联CAR-T细胞可作为一种策略，用于克服卵巢癌和胰腺癌中的肿瘤细胞异质性，并可能有助于设计基于其一次一种抗原结合特性以及基于抗原密度的抗原驱动的肿瘤细胞杀伤作用的治疗方法。
