Characterization of CAR T Cells Manufactured Using Genetically Engineered Artificial Antigen Presenting Cells

Document Type : Original Article


1 Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

2 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

3 Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran

4 Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

5 Department of Anatomical Sciences, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

6 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

7 Advanced Therapy Medicinal Product Technology Development Center (ATMP-TDC), Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran


Objective: 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.
Materials and Methods: 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.
Results: 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.
Conclusion: 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.


Main Subjects

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