Sayadmanesh Ali, Azadbakht Mohamad, Yari Kheirollah, Abedelahi Ali, Shafaei Hajar, Shanehbandi Dariush, Baradaran Behzad, Basiri Mohsen
Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Cell J. 2023 Oct 9;25(10):674-687. doi: 10.22074/cellj.2023.2001712.1304.
Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for the treatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is an important concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansion is to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 for T cell activation. The aim of this study was to characterize CAR T cells generated using this aAPC-mediated approach in terms of expansion efficiency, immunophenotype, and cytotoxicity.
In this experimental study, we generated an aAPC line by engineering K562 cells to express a membrane-bound anti-CD3 (mOKT3). T cell activation was performed by co-culturing PBMCs with either mitomycin C-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CARtransduced T cells were characterized in terms of expansion, activation markers, interferon gamma (IFN-γ) secretion, CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCs and antibodies were also investigated using a bioluminescence-based co-culture assay.
Our findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to that using the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memory T cells in the final product, we did not observe a significant difference in IFN-γ secretion, cytotoxic activity or exhaustion between CAR T cells generated with aAPC or antibodies.
Our results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR T cells, both methods can be used to manufacture potent CAR T cells. These findings are instrumental for the improvement of the CAR T cell manufacturing process and future applications of aAPC-mediated expansion of CAR T cells.
嵌合抗原受体（CAR）T细胞疗法最近已成为治疗不同类型癌症的一种有前景的方法。在成本和产品质量方面改进CAR T细胞制造是扩大该疗法可及性的一个重要关注点。一种提高T细胞扩增的提议策略是使用表达膜结合抗CD3用于T细胞激活的基因工程人工抗原呈递细胞（aAPC）。本研究的目的是从扩增效率、免疫表型和细胞毒性方面对使用这种aAPC介导方法产生的CAR T细胞进行表征。
在本实验研究中，我们通过对K562细胞进行工程改造以表达膜结合抗CD3（mOKT3）来生成一个aAPC系。通过将外周血单核细胞（PBMC）与丝裂霉素C处理的aAPC或表面固定的抗CD3和抗CD28抗体共培养来进行T细胞激活。未转导和CD19 - CAR转导的T细胞在扩增、激活标志物、γ干扰素（IFN - γ）分泌、CD4/CD8比值、记忆表型和耗竭标志物方面进行表征。还使用基于生物发光的共培养测定法研究了由aAPC和抗体产生的CD19 - CAR T细胞的细胞毒性。
我们的研究结果表明，工程改造的aAPC系具有与基于抗体的方法类似的扩增CAR T细胞的潜力。尽管用aAPC激活导致最终产物中CD8⁺和效应记忆T细胞的比例更高，但我们未观察到用aAPC或抗体产生的CAR T细胞在IFN - γ分泌、细胞毒性活性或耗竭方面存在显著差异。
我们的结果表明，尽管aAPC和基于抗体的CAR T细胞的免疫表型存在差异，但两种方法均可用于制造有效的CAR T细胞。这些发现有助于改进CAR T细胞制造过程以及aAPC介导的CAR T细胞扩增的未来应用。
