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

Document Type : Original Article

Authors

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

Abstract

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.

Keywords

Main Subjects

References

  1. Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science. 2015; 348(6230): 62- 68.
  2. Redeker A, Arens R. Improving adoptive T cell therapy: the particular role of T cell costimulation, cytokines, and post-transfer vaccination. Front Immunol. 2016; 7: 345.
  3. Maus MV, Fraietta JA, Levine BL, Kalos M, Zhao Y, June CH. Adoptive immunotherapy for cancer or viruses. Annu Rev Immunol. 2014; 32: 189-225.
  4. Mullard A. FDA approves first CAR T therapy. Nat Rev Drug Discov. 2017; 16(10): 669.
  5. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006; 314(5796): 126-129.
  6. Noonan KA, Huff CA, Davis J, Lemas MV, Fiorino S, Bitzan J, et al. Adoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma. Sci Transl Med. 2015; 7(288): 288ra78.
  7. Xu D, Jin G, Chai D, Zhou X, Gu W, Chong Y, et al. The development of CAR design for tumor CAR-T cell therapy. Oncotarget. 2018; 9(17): 13991-14004.
  8. June CH, Sadelain M. Chimeric antigen receptor therapy. N Engl J Med. 2018; 379(1): 64-73.
  9. Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021; 11(4): 69.
  10. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. CAR-T cell therapy in T-cell malignancies: is success a low-hanging fruit? Stem Cell Res Ther. 2021; 12(1): 527.
  11. Iyer RK, Bowles PA, Kim H, Dulgar-Tulloch A. Industrializing autologous adoptive immunotherapies: manufacturing advances and challenges. Front Med (Lausanne). 2018; 5: 150.
  12. McCarron A, Donnelley M, McIntyre C, Parsons D. Challenges of up-scaling lentivirus production and processing. J Biotechnol. 2016; 240: 23-30.
  13. Merten OW, Hebben M, Bovolenta C. Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016; 3: 16017.
  14. Schmidts A, Marsh LC, Srivastava AA, Bouffard AA, Boroughs AC, Scarfò I, et al. Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources. J Immunother Cancer. 2020; 8(2): e000990.
  15. Thomas AK, Maus MV, Shalaby WS, June CH, Riley JL. A cellbased artificial antigen-presenting cell coated with anti-CD3 and CD28 antibodies enables rapid expansion and long-term growth of CD4 T lymphocytes. Clin Immunol. 2002; 105(3): 259-272.
  16. Maus MV, Thomas AK, Leonard DG, Allman D, Addya K, Schlienger K, et al. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat Biotechnol. 2002; 20(2): 143-148.
  17. Prakken B, Wauben M, Genini D, Samodal R, Barnett J, Mendivil A, et al. Artificial antigen-presenting cells as a tool to exploit the immune ‘synapse’. Nat Med. 2000; 6(12): 1406-1410.
  18. Borrello IM, Levitsky HI, Stock W, Sher D, Qin L, DeAngelo DJ, et al. Granulocyte-macrophage colony-stimulating factor (GM-CSF)- secreting cellular immunotherapy in combination with autologous stem cell transplantation (ASCT) as postremission therapy for acute myeloid leukemia (AML). Blood. 2009; 114(9): 1736-1745.
  19. Qin L, Smith BD, Tsai HL, Yaghi NK, Neela PH, Moake M, et al. Induction of high-titer IgG antibodies against multiple leukemia-associated antigens in CML patients with clinical responses to K562/ GVAX immunotherapy. Blood Cancer J. 2013; 3(9): e145.
  20. Yekehfallah V, Pahlavanneshan S, Sayadmanesh A, Momtahan Z, Ma B, Basiri M. Generation and functional characterization of PLAP CAR-T cells against cervical cancer cells. Biomolecules. 2022; 12(9): 1296.
  21. Want MY, Bashir Z, Najar RA. T cell based immunotherapy for cancer: approaches and strategies. Vaccines (Basel). 2023; 11(4): 835.
  22. Sheykhhasan M, Manoochehri H, Dama P. Use of CAR T-cell foracute lymphoblastic leukemia (ALL) treatment: a review study. Cancer Gene Ther. 2022; 29(8-9): 1080-1096.
  23. Houot R, Schultz LM, Marabelle A, Kohrt H. T-cell-based Immunotherapy: adoptive cell transfer and checkpoint inhibition. Cancer Immunol Res. 2015; 3(10): 1115-1122.
  24. Ramos CA, Heslop HE, Brenner MK. CAR-T cell therapy for lymphoma. Annu Rev Med. 2016; 67: 165-183.
  25. Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med. 1973; 137(5): 1142-1162.
  26. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991; 9: 271-296.
  27. Inaba K, Metlay JP, Crowley MT, Steinman RM. Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J Exp Med. 1990; 172(2): 631-640.
  28. Ye K, Li F, Wang R, Cen T, Liu S, Zhao Z, et al. An armed oncolytic virus enhances the efficacy of tumor-infiltrating lymphocyte therapy by converting tumors to artificial antigen-presenting cells in situ. Mol Ther. 2022; 30(12): 3658-3676.
  29. Ratta M, Fagnoni F, Curti A, Vescovini R, Sansoni P, Oliviero B, et al. Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6. Blood. 2002; 100(1): 230-237. 30. Satthaporn S, Robins A, Vassanasiri W, El-Sheemy M, Jibril JA, Clark D, et al. Dendritic cells are dysfunctional in patients with operable breast cancer. Cancer Immunol Immunother. 2004; 53(6): 510-518.
  30. Neal LR, Bailey SR, Wyatt MM, Bowers JS, Majchrzak K, Nelson MH, et al. The basics of artificial antigen presenting cells in T cellbased cancer immunotherapies. J Immunol Res Ther. 2017; 2(1): 68-79.
  31. Wherry EJ. T cell exhaustion. Nat Immunol. 2011; 12(6): 492-499.
  32. Butler MO, Imataki O, Yamashita Y, Tanaka M, Ansén S, Berezovskaya A, et al. Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help. PLoS One. 2012; 7(1): e30229.
  33. Shrestha B, Zhang Y, Yu B, Li G, Boucher JC, Beatty NJ, et al. Generation of antitumor T cells for adoptive cell therapy with artificial antigen presenting cells. J Immunother. 2020; 43(3): 79-88.
  34. Vidán MT, Fernández-Gutiérrez B, Hernández-García C, Serra JA, Ribera JM, Pérez-Blas M, et al. Functional integrity of the CD28 co-stimulatory pathway in T lymphocytes from elderly subjects. Age Ageing. 1999; 28(2): 221-227.
  35. Beyersdorf N, Kerkau T, Hünig T. CD28 co-stimulation in T-cell homeostasis: a recent perspective. Immunotargets Ther. 2015; 4: 111-122.
  36. Kawalekar OU, O’Connor RS, Fraietta JA, Guo L, McGettigan SE, Posey AD Jr, et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity. 2016; 44(2): 380-390.

Files

Share

How to cite

Statistics