Advances in Clinical and Experimental Medicine

Adv Clin Exp Med
Impact Factor (IF) – 1.514
Index Copernicus (ICV 2018) – 157.72
MNiSW – 40
Average rejection rate – 84.38%
ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
Periodicity – monthly

Download PDF

Advances in Clinical and Experimental Medicine

2019, vol. 28, nr 2, February, p. 159–164

doi: 10.17219/acem/90772

Publication type: original article

Language: English

Download citation:

  • BIBTEX (JabRef, Mendeley)
  • RIS (Papers, Reference Manager, RefWorks, Zotero)

Creative Commons BY-NC-ND 3.0 Open Access

A quantitative method for measuring the transfection efficiency of CD19-directed chimeric antigen receptor in target cells

Na Fang1,B,C, Niannian Zhong1,C, Tingxuan Gu1,B, Yahui Wang1,B, Xiangqian Guo1,E, Shaoping Ji1,A,D,F

1 Department of Biochemistry and Molecular Biology, Medical School, Henan University, Kaifeng, China

Abstract

Background. Adoptive cell therapy (ACT) based on chimeric antigen receptors (CARs) expressed on the surface of T cells shows a remarkable clinical outcome, particularly for B-cell malignancies. However, toxicity and side effects of CD19-redirected CAR T cells have been observed concurrently in most cases due to cytokine release and tumor cell lysis. Therefore, strictly controlling the amount of valid T cells re-transfused to patients seems to be an important step in reducing toxicity and side effects of CAR T cells. Transfection efficiency via lentiviral particles varies widely in different cases.
Objectives. The aim of this study was to accurately calculate and control the number of valid CAR T cells through ACT because the restriction antibiotics gene or the fluorescence gene are not suitable for tracking or screening for valid transfected T cells.
Material and Methods. We expressed and purified a GFP-CD19 fusion protein as a probe to measure the expression efficiency of CD19-redirected CAR on the cell surface in adherent and suspension cell lines.
Results. We can precisely calculate the transfected efficiency of lentiviral particles by counting the number of GFP-labeled cells under a microscope, as well as calculate the percentage by comparing the number of GFP-labeled cells to total cells.
Conclusion. We propose a method to control the number of valid cells in ACT and to reduce toxicity and side effects in clinical use – a convenient technique for monitoring the dosage of CAR T cells for patients.

Key words

immunotherapy, CAR-T, CD19, examination, B-cell malignancy

References (29)

  1. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A. 1989;86(24):10024–10028.
  2. Huston JS, Levinson D, Mudgett-Hunter M, et al. Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988;85(16):5879–5883.
  3. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365(8):725–733.
  4. Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368(16):1509–1518.
  5. Scheuermann RH, Racila E. CD19 antigen in leukemia and lymphoma diagnosis and immunotherapy. Leuk Lymphoma. 1995;18(5–6):385–397.
  6. Depoil D, Fleire S, Treanor BL, et al. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand. Nat Immunol. 2008;9(1):63–72.
  7. Brentjens RJ, Davila ML, Riviere I, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177):177ra38.
  8. Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med. 2011;3(95):95ra73.
  9. Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood. 2010;116(20):4099–4102.
  10. Posey AD Jr, Schwab RD, Boesteanu AC, et al. Engineered CAR T cells targeting the cancer-associated Tn-glycoform of the membrane mucin MUC1 control adenocarcinoma. Immunity. 2016;44(6):1444–1454.
  11. Wang X, Riviere I. Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies. Cancer Gene Ther. 2015;22(2):85–94.
  12. Davila ML, Sadelain M. Biology and clinical application of CAR T cells for B cell malignancies. Int J Hematol. 2016;104(1):6–17.
  13. Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood. 2012;119(12):2709–2720.
  14. Johnson FH, Shimomura O, Saiga Y. Action of cyanide on Cypridina luciferin. J Cell Comp Physiol. 1962;59:265–272.
  15. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994;263(5148):802–805.
  16. Arun KH, Kaul CL, Ramarao P. Green fluorescent proteins in receptor research: An emerging tool for drug discovery. J Pharmacol Toxicol Methods. 2005;51(1):1–23.
  17. Subedi GP, Satoh T, Hanashima S, et al. Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector. Protein Expr Purif. 2012;82(1):197–204.
  18. Turtle CJ, Riddell SR, Maloney DG. CD19-Targeted chimeric antigen receptor-modified T-cell immunotherapy for B-cell malignancies. Clin Pharmacol Ther. 2016;100(3):252–258.
  19. Onea AS, Jazirehi AR. CD19 chimeric antigen receptor (CD19 CAR)-redirected adoptive T-cell immunotherapy for the treatment of relapsed or refractory B-cell Non-Hodgkin’s Lymphomas. Am J Cancer Res. 2016;6(2):403–424.
  20. Sadelain M, Brentjens R, Riviere I, Park J. CD19 CAR therapy for acute lymphoblastic leukemia. Am Soc Clin Oncol Educ Book. 2015:e360–363.
  21. Sadelain M. CAR therapy: The CD19 paradigm. J Clin Invest. 2015;125(9):3392–3400.
  22. Miller BC, Maus MV. CD19-targeted CAR T cells: A new tool in the fight against B cell malignancies. Oncol Res Treat. 2015;38(12):683–690.
  23. Romanski A, Uherek C, Bug G, et al. CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies. J Cell Mol Med. 2016;20(7):1287–1294.
  24. Sabatino M, Hu J, Sommariva M, et al. Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies. Blood. 2016;128(4):519–528.
  25. Kebriaei P, Singh H, Huls MH, et al. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J Clin Invest. 2016;126(9):3363–3376.
  26. Ruella M, Barrett DM, Kenderian SS, et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunother-apies. J Clin Invest. 2016;126(10):3814–3826.
  27. Wang L, Ma N, Okamoto S, et al. Efficient tumor regression by adoptively transferred CEA-specific CAR-T cells associated with symptoms of mild cytokine release syndrome. Oncoimmunology. 2016;5(9):e1211218.
  28. Ansari AM, Ahmed AK, Matsangos AE, et al. Cellular GFP toxicity and immunogenicity: Potential confounders in in vivo cell tracking experi-ments. Stem Cell Rev. 2016;12(5):553–559.
  29. Jawale CV, Lee JH. Development of a biosafety enhanced and immunogenic Salmonella enteritidis ghost using an antibiotic resistance gene free plasmid carrying a bacteriophage lysis system. PLoS One. 2013;8(10):e78193.