Majumdar Shubhabrata, Echelibe Hilda, Bettini Maria, Bettini Matthew L
Immunology Graduate Program, Baylor College of Medicine, Houston, TX, United States.
Department of Pathology, University of Utah, Salt Lake City, UT, United States.
Front Immunol. 2025 Jan 16;15:1509980. doi: 10.3389/fimmu.2024.1509980. eCollection 2024.
Chimeric antigen receptor (CAR) expressing T-cells have shown great promise for the future of cancer immunotherapy with the recent clinical successes achieved in treating different hematologic cancers. Despite these early successes, several challenges remain in the field that require to be solved for the therapy to be more efficacious. One such challenge is the lack of long-term persistence of CD28 based CAR T-cells in patients. Although, CD28 based CAR T-cells elicit a robust acute anti-tumor response, they are more prone to early exhaustion, terminal differentiation and cell death due to their strong signaling patterns. Hence attenuation of signaling strength in CD28 based CARs is an accepted strategy to improve long-term CAR T-cell function and persistence in patients. Previous studies with the conventional T-cell receptor (TCR) have suggested that manipulation of CD3 immunoreceptor tyrosine-based activation motif (ITAM) sequences can alter TCR signaling strength. Based on these studies, we have designed 2 generation murine anti-CD19 CD28 based CARs with restricted CD3ζ ITAM sequence diversity while maintaining a multiplicity of three. They are called ζAAA, ζBBB and ζCCC based on which CD3ζ ITAM they express. The goal of the study is to understand the non-redundant signaling properties of the individual CD3ζ ITAMs and their effect on CAR T-cell function. We hypothesized that the individual CD3ζ ITAMs will exhibit unique signaling properties in the ITAM restricted CARs which may allow for optimization of CAR signaling and improve CAR T-cell persistence and function.
We subjected the ITAM restricted CAR T cells to various conditions of in vitro stimulation using CD19+ tumor cells or CD19-coated magnetic beads. Immunoblotting and flow cytometry based Ca2+ signaling assays were used to quantify signaling differences. Functional differences were studied using in vitro cytotoxicity, degranulation and cytokine expression assays. CAR T cell exhaustion and differentiation were studied using an in vitro exhaustion assay.
We observed that ζAAA CARs had stronger signaling strength compared to ζBBB and ζCCC CARs. The signaling differences were reflected in their functional activation profiles with T-cells expressing ζAAA CARs having a strong activation profile and ζCCC CARs having a weak activation profile. ζCCC CAR T cells were less prone to differentiation and exhaustion.
Since, weaker signaling ζCCC CARs favored less cell death, exhaustion and differentiation, they might be better candidates for improving long term survival and persistence of CAR T cells in patients.
表达嵌合抗原受体（CAR）的T细胞在癌症免疫治疗领域展现出了巨大的前景，近期在治疗不同血液系统癌症方面取得了临床成功。尽管取得了这些早期成功，但该领域仍存在一些挑战，需要解决才能使治疗更有效。其中一个挑战是基于CD28的CAR T细胞在患者体内缺乏长期持久性。虽然基于CD28的CAR T细胞能引发强烈的急性抗肿瘤反应，但由于其强烈的信号模式，它们更容易早期耗竭、终末分化和细胞死亡。因此，减弱基于CD28的CARs中的信号强度是一种公认的策略，以改善CAR T细胞在患者体内的长期功能和持久性。先前对传统T细胞受体（TCR）的研究表明，操纵CD3免疫受体酪氨酸基激活基序（ITAM）序列可以改变TCR信号强度。基于这些研究，我们设计了两代基于鼠抗CD19 CD28的CARs，其CD3ζ ITAM序列多样性受限，同时保持三聚体形式。根据它们表达的CD3ζ ITAM，分别称为ζAAA、ζBBB和ζCCC。本研究的目的是了解各个CD3ζ ITAMs 的非冗余信号特性及其对CAR T细胞功能的影响。我们假设，在ITAM受限的CARs中，各个CD3ζ ITAMs将表现出独特的信号特性，这可能有助于优化CAR信号并改善CAR T细胞的持久性和功能。
我们使用CD19 +肿瘤细胞或CD19包被的磁珠对ITAM受限的CAR T细胞进行各种体外刺激条件处理。采用免疫印迹和基于流式细胞术的Ca2 +信号测定法来量化信号差异。使用体外细胞毒性、脱颗粒和细胞因子表达测定法研究功能差异。使用体外耗竭测定法研究CAR T细胞的耗竭和分化。
我们观察到，与ζBBB和ζCCC CARs相比，ζAAA CARs具有更强的信号强度。信号差异反映在它们的功能激活谱中，表达ζAAA CARs的T细胞具有强烈的激活谱，而表达ζCCC CARs的T细胞具有较弱的激活谱。ζCCC CAR T细胞较不容易分化和耗竭。
由于信号较弱的ζCCC CARs导致较少的细胞死亡、耗竭和分化，它们可能是改善CAR T细胞在患者体内长期存活和持久性的更好候选者。
