Coren Lori V, Trivett Matthew T, Welker Jorden L, Thomas James A, Gorelick Robert J, Kose Emek, Immonen Taina T, Czarra Kelli, Fennessey Christine M, Trubey Charles M, Lifson Jeffrey D, Swanstrom Adrienne E
AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
PLoS One. 2025 Jan 9;20(1):e0314751. doi: 10.1371/journal.pone.0314751. eCollection 2025.
T cell immunotherapy success is dependent on effective levels of antigen receptor expressed at the surface of engineered cells. Efforts to optimize surface expression in T cell receptor (TCR)-based therapeutic approaches include optimization of cellular engineering methods and coding sequences, and reducing the likelihood of exogenous TCR α and β chains mispairing with the endogenous TCR chains. Approaches to promote correct human TCR chain pairing include constant region mutations to create an additional disulfide bond between the two chains, full murinization of the constant region of the TCR α and β sequences, and a minimal set of murine mutations to the TCR α and β constant regions. Preclinical animal models are valuable tools to optimize engineering designs and methods, and to evaluate the potential for off-target tissue injury. To further develop rhesus macaque models for TCR based cellular immunotherapy, we tested methods for improving cell surface expression of rhesus macaque TCR in rhesus macaque primary cells by generating five alternative TCRαβ constant region constructs in the context of a SIV Gag-specific TCR: 1. human codon optimized rhesus macaque (RH); 2. RH TCR with an additional disulfide linkage; 3. rhesus macaque constant sequences with minimal murine amino acid substitutions; 4. murinized constant sequences; and 5. murinized constant sequences with a portion of the exposed FG loop in the β constant sequence replaced with rhesus macaque sequence to reduce potential immunogencity. Murinization or mutation of a minimal set of amino acids to the corresponding murine sequence of the constant region resulted in the greatest increase in rhesus macaque TCR surface expression relative to wild type. All novel TCR constructs retained the ability to induce production of cytokines in response to cognate peptide antigen specific stimulation. This work can inform the design of TCRs selected for use in rhesus macaque models of TCR-based cellular immunotherapy.
T细胞免疫疗法的成功取决于工程细胞表面表达的抗原受体达到有效水平。在基于T细胞受体（TCR）的治疗方法中，优化表面表达的努力包括优化细胞工程方法和编码序列，以及降低外源性TCRα和β链与内源性TCR链错配的可能性。促进正确的人类TCR链配对的方法包括恒定区突变以在两条链之间形成额外的二硫键、TCRα和β序列恒定区的完全鼠源化，以及对TCRα和β恒定区进行最少的鼠源突变。临床前动物模型是优化工程设计和方法以及评估脱靶组织损伤可能性的宝贵工具。为了进一步开发基于TCR的细胞免疫疗法的恒河猴模型，我们通过在SIV Gag特异性TCR的背景下生成五种替代的TCRαβ恒定区构建体，测试了改善恒河猴原代细胞中恒河猴TCR细胞表面表达的方法：1. 人类密码子优化的恒河猴（RH）；2. 具有额外二硫键的RH TCR；3. 具有最少鼠源氨基酸替代的恒河猴恒定序列；4. 鼠源化恒定序列；5. 鼠源化恒定序列，其中β恒定序列中一部分暴露的FG环被恒河猴序列取代以降低潜在免疫原性。相对于野生型，将恒定区的最少一组氨基酸鼠源化或突变为相应的鼠源序列导致恒河猴TCR表面表达增加最多。所有新型TCR构建体在受到同源肽抗原特异性刺激时均保留诱导细胞因子产生的能力。这项工作可为选择用于基于TCR的细胞免疫疗法的恒河猴模型的TCR设计提供参考。
