Early cytokine and chemokine signals shape the anti-AML activity of bispecific engager-secreting T cells

Immunotherapies, including cell therapies, are effective anti-cancer agents. However, cellular product persistence can be limiting with short functional duration of activity contributing to disease relapse. A variety of manufacturing protocols are used to generate therapeutic engineered T-cells; these differ in techniques used for T-cell isolation, activation, genetic modification, and other methodology. We sought to determine how preselection affected the phenotype of T cells engineered to secrete a CD123xCD3 bispecific engager (ENG-T). These cells were designed to treat acute myeloid leukemia (AML). We evaluated the effect of T-cell selection on transduction efficiency, T-cell activation, short- and long-term anti-AML cytotoxicity, and gene transcription. Unselected, CD4, CD8, and CD4/CD8 pre-selected ENG-T cells have minor differences in T-cell subset components, equivalent activation, and equal cytotoxicity in short-term assays. While unselected and CD4/CD8-selected ENG-T cells have identical CD4:CD8 composition prior to target cell exposure, serial stimulation in vitro showed CD4/CD8 pre-selection supports ENG-T cell survival and long-term activity. Likewise, CD4 and CD4/CD8 pre-selected ENG-T cells display superior anti-tumor efficacy and prolong murine survival in AML xenografts. Unselected ENG-T cells are exposed to cytokines during early manufacture that imprint upregulation of intracellular inflammatory pathways. This early activation likely underpins long-term observed functional differences. Pre-selection of T cells from banked patient biospecimens decreased blast contamination, exposure to inflammatory cytokines, and may improve T-cell expansion during manufacture. Pre-selection of T-cell products should continue to be performed to enhance the quality of clinical cellular therapeutics. Bulk RNAseq was performed on T cells engineered to secrete a bispecific engager molecule. Engineered T cells were manufactured either from CD4/CD8-selected starting material or directly from whole peripheral blood mononuclear cells. Cells from 3 healthy donors were expanded and frozen on day 7 after activation for processing.

包括细胞疗法在内的免疫治疗是有效的抗癌手段。然而细胞产品的存续性常受限于较短的功能持续时长，这会导致疾病复发。目前存在多种生产方案用于制备治疗性工程化T细胞（engineered T-cell），不同方案在T细胞分离、活化、基因修饰及其他操作技术上存在差异。本研究旨在探究预分选对分泌CD123xCD3双特异性接合蛋白的工程化T细胞（ENG-T）表型的影响，该类细胞被设计用于治疗急性髓系白血病（acute myeloid leukemia, AML）。我们评估了T细胞分选对转导效率、T细胞活化、短期及长期抗AML细胞毒性以及基因转录的影响。未分选组、CD4分选组、CD8分选组以及CD4/CD8预分选组的ENG-T细胞在T细胞亚群组成、活化水平及短期试验中的细胞毒性上仅存在细微差异。尽管在靶细胞暴露前，未分选组与CD4/CD8分选组的ENG-T细胞CD4:CD8比例完全一致，但体外连续刺激实验显示，CD4/CD8预分选可维持ENG-T细胞的存活与长期活性。同样，CD4分选组及CD4/CD8预分选组的ENG-T细胞展现出更优的抗肿瘤效果，并可延长AML异种移植模型小鼠的存活时间。未分选的ENG-T细胞在早期生产过程中会暴露于细胞因子环境，这会诱导细胞内炎症通路的上调表达。这种早期活化或许是长期观察到的功能差异的关键成因。从储存的患者生物样本中预分选T细胞，可减少母细胞污染、降低细胞因子暴露风险，并可能提升生产过程中T细胞的扩增效率。为提升临床细胞治疗产品的质量，T细胞产品的预分选流程应当继续推广应用。本研究对分泌双特异性接合蛋白的工程化T细胞进行了批量RNA测序（bulk RNAseq）。工程化T细胞的制备分别以CD4/CD8分选后的起始材料，或直接以全外周血单个核细胞为原料进行。来自3名健康供者的T细胞在活化后第7天进行扩增并冻存，以待后续处理。