嵌合抗原受体（CAR）T细胞疗法已经彻底改变了治疗血液恶性肿瘤的方式，但由于在肿瘤微环境中缺乏持久性和功能，尚未在实体肿瘤中获得类似的成功。我们先前报道了通过分泌抗PD-1 scFv增强CAR T细胞疗法在工程实体肿瘤模型中的效果，通过增强CAR T细胞的抗肿瘤功效，扩张和活力得到了证实。此后，我们改进了这个平台，创造了一个更优秀的细胞产品 - 分泌抗PD-1 scFv融合的单链三聚体4-1BB配体（αPD1-41BBL）的CAR T细胞。 4-1BB信号促进细胞毒性T淋巴细胞的增殖和生存能力，但在临床上使用激动剂抗体靶向4-1BB一直受到了低的抗肿瘤活性和高毒性的限制。使用4-1BB内酰胺结构域作为协同刺激信号的CAR T细胞，与使用CD28内酰胺结构域协同刺激的CAR T细胞相比，表现出较轻的抗肿瘤反应和更长的持久性。我们首次将CD28协同刺激的CAR T细胞改造成分泌融合蛋白的细胞，其中包含可溶的三聚体4-1BB配体。体外和体内实验证明，CAR19.αPD1-41BBL T细胞表现出减少的抑制性受体上调、增强的持久性和增殖能力，并且具有较少分化的记忆状态，与没有额外4-1BB：4-1BBL协同刺激的CAR T细胞相比较。因此，接受CAR19.αPD1-41BBL T细胞治疗的小鼠显示出显著的肿瘤生长控制和整体生存率的改善。受我们以CD19为模型抗原的临床前成功的激励，我们生产了靶向介质膜素的CAR T细胞，并证实了αPD1-41BBL分泌的CAR T细胞的增强型实体肿瘤疗效。

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies but has yet to achieve similar success in solid tumors due to a lack of persistence and function in the tumor microenvironment. We previously reported the augmentation of CAR T cell therapy in an engineered solid tumor model through the secretion of anti-PD-1 scFv, as shown by enhanced CAR T cell antitumor efficacy, expansion, and vitality. We have since improved the platform to create a superior cellular product - CAR T cells secreting single-chain trimeric 4-1BB ligand fused to anti-PD-1 scFv (αPD1-41BBL). 4-1BB signaling promotes cytotoxic T lymphocyte proliferation and survival but targeting 4-1BB with agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity. CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder anti-tumor response and longer persistence compared to CAR T cells costimulated by CD28 endodomain. We have, for the first time, engineered CD28-costimulated CAR T cells to secrete a fusion protein containing the soluble trimeric 4-1BB ligand. In vitro and in vivo, CAR19.αPD1-41BBL T cells exhibited reduced inhibitory receptor upregulation, enhanced persistence and proliferation, and a less differentiated memory status compared to CAR T cells without additional 4-1BB:4-1BBL costimulation. Accordingly, CAR19.αPD1-41BBL T cell-treated mice displayed significantly improved tumor growth control and overall survival. Spurred on by our preclinical success targeting CD19 as a model antigen, we produced mesothelin-targeting CAR T cells and confirmed the enhanced solid tumor efficacy of αPD1-41BBL-secreting CAR T cells.