Smagghe Benoit J, Carter Mark G, Yi Kevin R, Nash Jac-Leen S S, Grant Trevor J, Miller Dan S, Moe Scott T, Smith David D, Nash Michael J, Miller Natalie K, Agarkov Laura L, Marquez Jacy P, Stewart Andrew K, Bamdad Cynthia C
Minerva Biotechnologies Corporation, Waltham, Massachusetts, USA.
Mercy Lab Foundation, Pasadena, California, USA.
J Immunother Cancer. 2025 May 30;13(5):e010577. doi: 10.1136/jitc-2024-010577.
We developed Chimeric antigen receptor (CAR) T cells targeting mucin 1 (MUC1)* (muk * (muk 1 star)), which is the tumor-associated growth factor receptor form of MUC1. Our antibody, MNC2, uniquely binds to MUC1* on cancer cells but does not bind to full-length MUC1, which is expressed on all normal epithelial cells. We tested the ability of the Tyr to Phe mutations in CD3ζ, known as "1XX", to increase in vivo persistence and enable the recognition and killing of low antigen-expressing cancer cells.
We performed in vivo experiments comparing CARs with either 4-1BB or CD28 co-stimulatory domains, with or without the "1XX" Tyr to Phe mutations in ITAMs 2 and 3 of the CD3ζ signaling domain. All CARs were targeted to the tumor using the same huMNC2-scFv. To explore the sensitivity of each CAR, tumors comprising varying percentages of high MUC1* expressing cancer cells were xenografted. Further, wild-type low MUC1* expressing cells were engineered to fluoresce red while the cells engineered to express more MUC1* were made to fluoresce green. This experimental design allowed us to compare the sensitivity limits of the CARs against low versus high antigen-expressing cancer cells.
At high dose, all the CAR T cells effectively killed high antigen-expressing tumors in the short term. However, only the CAR bearing the 1XX mutations inhibited tumor recurrence in long-term experiments. Interestingly, in animals treated with CARs bearing wild-type CD3ζ, tumor recurrence was driven by the low antigen-expressing cells. Only the CAR bearing 1XX mutations demonstrated the ability to kill low antigen-expressing tumors, even when administered at low dose. Post-sacrifice analysis showed that the CAR T cells with 1XX mutations persisted longer in vivo than either 4-1BB or CD28 CAR T cells with wild-type CD3ζ.
These results support that the combination of targeting MUC1*, the growth factor receptor form of MUC1, with a CAR T bearing the 1XX mutations in CD3ζ has therapeutic potential for the treatment of solid tumor cancers.
我们研发了靶向粘蛋白1（MUC1）（muk （muk 1星号））的嵌合抗原受体（CAR）T细胞，MUC1是MUC1的肿瘤相关生长因子受体形式。我们的抗体MNC2特异性结合癌细胞上的MUC1，但不结合所有正常上皮细胞上表达的全长MUC1。我们测试了CD3ζ中酪氨酸（Tyr）到苯丙氨酸（Phe）的突变（即“1XX”）增加体内持久性以及识别和杀伤低抗原表达癌细胞的能力。
我们进行了体内实验，比较具有4-1BB或CD28共刺激结构域的CAR，以及在CD3ζ信号结构域的免疫受体酪氨酸激活基序（ITAM）2和3中有无“1XX”Tyr到Phe突变的CAR。所有CAR均使用相同的huMNC2单链抗体片段（scFv）靶向肿瘤。为了探究每种CAR的敏感性，移植了含有不同比例高表达MUC1癌细胞的肿瘤。此外，将野生型低表达MUC1的细胞改造为发出红色荧光，而将改造后表达更多MUC1*的细胞改造为发出绿色荧光。这种实验设计使我们能够比较CAR对低抗原表达癌细胞与高抗原表达癌细胞的敏感性极限。
在高剂量时，所有CAR T细胞在短期内均能有效杀伤高抗原表达肿瘤。然而，在长期实验中，只有带有1XX突变的CAR抑制了肿瘤复发。有趣的是，在用野生型CD3ζ的CAR治疗的动物中，肿瘤复发是由低抗原表达细胞驱动的。只有带有1XX突变的CAR表现出杀伤低抗原表达肿瘤的能力，即使在低剂量给药时也是如此。处死动物后的分析表明，带有1XX突变的CAR T细胞在体内的持续时间比带有野生型CD3ζ的4-1BB或CD28 CAR T细胞更长。
这些结果支持，靶向MUC1的生长因子受体形式MUC1*与在CD3ζ中带有1XX突变的CAR T细胞相结合，对实体肿瘤癌症具有治疗潜力。
