Production of CD87 Antigen-Specific CAR-T Lymphocytes and Assessment of Their In Vitro Functional Activity

M.V. Neklesova, Sergei Vladimirovich Smirnov, A.A. Shatilova, K.A. Levchuk, A.E. Ershova, S.A. Silonov,

DOI:

https://doi.org/10.21320/2500-2139-2022-15-4-340-348

Aim. To generate anti-CD87 CAR-T lymphocytes and to assess their in vitro functional activity.

Materials & Methods. Т-lymphocytes isolated from healthy donor peripheral blood were transduced with the anti-CD87-CAR, T2A, and FusionRed gene coding lentiviral vector. Transduction efficacy assessed by reporter protein FusionRed signal, subpopulation structure, and functional status of CAR-T lymphocytes were determined by flow cytometry. Interferon-γ (IFN-γ) expression by CAR-T lymphocytes was analyzed using immunoassay. Cytotoxic activity of CAR-T lymphocytes was evaluated during their co-cultivation with HeLa target cells by means of xCELLigence real-time assay.

Results. The efficacy of T-lymphocyte transduction was 8.4 %. The obtained CAR-T cells contained the markers of both CD27 and/or CD28 activation (92.91 % cases) and PD1 exhaustion (20.66 % cases). The population of CAR-T lymphocytes showed 98.51 % central memory T-cell phenotype and CD4/CD8 ratio of 1:7. IFN-γ concentration in the medium after co-cultivation of CAR-T lymphocytes with target cells appeared to be significantly higher than in control samples. The study demonstrates that generated CAR-T lymphocytes manifest specific cytotoxicity towards target cells with both unmodified expression and overexpression of CD87 antigen in HeLa cell lines. Cytotoxicity proved to be more pronounced with respect to the cell line with CD87 antigen overexpression.

Conclusion. Despite overexpression of PD1 exhaustion marker, CAR-T lymphocytes showed specific IFN-γ secretion and pronounced cytotoxic activity in interaction with CD87 antigen on target cell membranes. Therefore, anti-CD87 CAR-T lymphocytes can be applied in the treatment of hematologic as well as solid tumors. Since the observed difference in cytotoxicity does not linearly correlate with CD87 antigen density on the surface of attacked cells, the in vivo administration of a CAR-T cell drug should be designed to prevent cytotoxic risk for CD87-expressing healthy cells.

  • M.V. Neklesova Center for Personalized Medicine, VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; НЦМУ «Центр персонализированной медицины», ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
  • Sergei Vladimirovich Smirnov Center for Personalized Medicine, VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; НЦМУ «Центр персонализированной медицины», ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
  • A.A. Shatilova Center for Personalized Medicine, VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; НЦМУ «Центр персонализированной медицины», ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
  • K.A. Levchuk Center for Personalized Medicine, VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; НЦМУ «Центр персонализированной медицины», ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
  • A.E. Ershova Center for Personalized Medicine, VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; НЦМУ «Центр персонализированной медицины», ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
  • S.A. Silonov Center for Personalized Medicine, VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; НЦМУ «Центр персонализированной медицины», ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
  1. Stoppelli MP, Corti A, Soffientini A, et al. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci USA. 1985;82(15):4939–43. doi: 10.1073/pnas.82.15.4939. DOI: https://doi.org/10.1073/pnas.82.15.4939
  2. Behrendt N, Ronne E, Dano K. The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface. Biol Chem Hoppe Seyler. 1995;376(5):269–79.
  3. Кугаевская Е.В., Гуреева Т.А., Тимошенко О.С., Соловьева Н.И. Система активатора плазминогена урокиназного типа в норме и при жизнеугрожающих процессах (обзор). Общая реаниматология. 2018;14(6):61–79. doi: 10.15360/1813-9779-2018-6-61-79.
  4. [Kugaevskaya EV, Gureeva TA, Timoshenko OS, Solovyeva NI. Urokinase-type plasminogen activator system in norm and in life-threatening processes (Review). General Reanimatology. 2018;14(6):61–79. doi: 10.15360/1813-9779-2018-6-61-79. (In Russ)] DOI: https://doi.org/10.15360/1813-9779-2018-6-61-79
  5. Mahmood N, Mihalcioiu C, Rabbani SA. Multifaceted Role of the Urokinase-Type Plasminogen Activator (uPA) and Its Receptor (uPAR): Diagnostic, Prognostic, and Therapeutic Applications. Front Oncol. 2018;8(2):8–24. doi: 10.3389/fonc.2018.00024. DOI: https://doi.org/10.3389/fonc.2018.00024
  6. Alfano D, Gorrasi A, Li Santi A, et al. Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells. J Cell Mol Med. 2015;19(9):2262–72. doi: 10.1111/jcmm.12617. DOI: https://doi.org/10.1111/jcmm.12617
  7. Smith HW, Marshall CJ. Regulation of cell signalling by uPAR. Nat Rev Mol Cell Biol. 2010;11(1):23–36. doi: 10.1038/nrm2821. DOI: https://doi.org/10.1038/nrm2821
  8. Gorantla B, Asuthkar S, Rao JS, et al. Suppression of the uPAR-uPA system retards angiogenesis, invasion, and in vivo tumor development in pancreatic cancer cells. Mol Cancer Res. 2011;9(4):377–89. doi: 10.1158/1541-7786.MCR-10-0452. DOI: https://doi.org/10.1158/1541-7786.MCR-10-0452
  9. Amor C, Feucht J, Leibold J, et al. Senolytic CAR T cells reverse senescence-associated pathologies. Nature. 2020;583(7814):127–32. doi: 10.1038/s41586-020-2403-9. DOI: https://doi.org/10.1038/s41586-020-2403-9
  10. Kusch A, Gulba D. Die Bedeutung des uPA/uPAR-Systems fur die Entwicklung von Arteriosklerose und Restenose. Z Kardiol. 2001;90(1):307–18. doi: 10.1007/s003920170160. DOI: https://doi.org/10.1007/s003920170160
  11. Laurenzana A, Chilla A, Luciani C, et al. uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells. Int J Cancer. 2017;141(6):1190–200. doi: 10.1002/ijc.30817. DOI: https://doi.org/10.1002/ijc.30817
  12. Ahmad A, Kong D, Sarkar SH, et al. Inactivation of uPA and its receptor uPAR by 3,3’-diindolylmethane (DIM) leads to the inhibition of prostate cancer cell growth and migration. J Cell Biochem. 2009;107(3):516–27. doi: 10.1002/jcb.22152. DOI: https://doi.org/10.1002/jcb.22152
  13. Fox SB, Taylor M, Grondahl-Hansen J, et al. Plasminogen activator inhibitor-1 as a measure of vascular remodelling in breast cancer. J Pathol. 2001;195(2):236–43. doi: 10.1002/path.931. DOI: https://doi.org/10.1002/path.931
  14. Fisher JL, Field CL, Zhou H, et al. Urokinase plasminogen activator system gene expression is increased in human breast carcinoma and its bone metastases – a comparison of normal breast tissue, non-invasive and invasive carcinoma and osseous metastases. Breast Cancer Res Treat. 2000;61(1):1–12. doi: 10.1007/s10549-004-6659-9. DOI: https://doi.org/10.1007/s10549-004-6659-9
  15. Pierga JY, Bonneton C, Magdelenat H, et al. Real-time quantitative PCR determination of urokinase-type plasminogen activator receptor (uPAR) expression of isolated micrometastatic cells from bone marrow of breast cancer patients. Int J Cancer. 2005;114(2):291–8. doi: 10.1002/ijc.20698. DOI: https://doi.org/10.1002/ijc.20698
  16. Hildenbrand R, Schaaf A, Dorn-Beineke A, et al. Tumor stroma is the predominant uPA-, uPAR-, PAI-1-expressing tissue in human breast cancer: prognostic impact. Histol Histopathol. 2009;24(7):869–77. doi: 10.14670/HH-24.869.
  17. Boonstra MC, Verbeek FP, Mazar AP, et al. Expression of uPAR in tumor-associated stromal cells is associated with colorectal cancer patient prognosis: a TMA study. BMC Cancer. 2014;14:269. doi: 10.1186/1471-2407-14-269. DOI: https://doi.org/10.1186/1471-2407-14-269
  18. Graf M, Reif S, Hecht K, et al. High expression of urokinase plasminogen activator receptor (UPA-R) in acute myeloid leukemia (AML) is associated with worse prognosis. Am J Hematol. 2005;79(1):26–35. doi: 10.1002/ajh.20337. DOI: https://doi.org/10.1002/ajh.20337
  19. Plesner T, Ralfkiaer E, Wittrup M, et al. Expression of the receptor for urokinase-type plasminogen activator in normal and neoplastic blood cells and hematopoietic tissue. Am J Clin Pathol. 1994;102(6):835–41. doi: 10.1093/ajcp/102.6.835. DOI: https://doi.org/10.1093/ajcp/102.6.835
  20. Bene MC, Castoldi G, Knapp W, et al. CD87 (urokinase-type plasminogen activator receptor), function and pathology in hematological disorders: a review. Leukemia. 2004;18(3):394–400. doi: 10.1038/sj.leu.2403250. DOI: https://doi.org/10.1038/sj.leu.2403250
  21. Cummins KD, Gill S. Will CAR T cell therapy have a role in AML? Promises and pitfalls. Semin Hematol. 2019;56(2):155–63. doi: 10.1053/j.seminhematol.2018.08.008. DOI: https://doi.org/10.1053/j.seminhematol.2018.08.008
  22. Kramer MD, Spring H, Todd RF, et al. Urokinase-type plasminogen activator enhances invasion of human T cells (Jurkat) into a fibrin matrix. J Leukoc Biol. 1994;56(2):110–6. doi: 10.1002/jlb.56.2.110. DOI: https://doi.org/10.1002/jlb.56.2.110
  23. Bianchi E, Ferrero E, Fazioli F, et al. Integrin-dependent induction of functional urokinase receptors in primary T lymphocytes. J Clin Invest. 1996;98(5):1133–41. doi: 10.1172/JCI118896. DOI: https://doi.org/10.1172/JCI118896
  24. Xu Y, Zhang M, Ramos CA, et al. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood. 2014;123(24):3750–9. doi: 10.1182/blood-2014-01-552174. DOI: https://doi.org/10.1182/blood-2014-01-552174
  25. Sommermeyer D, Hudecek M, Kosasih PL, et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia. 2016;30(2):492–500. doi: 10.1038/leu.2015.247. DOI: https://doi.org/10.1038/leu.2015.247
  26. Baumeister SH, Murad J, Werner L, et al. Phase I Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients with AML/MDS and Multiple Myeloma. Cancer Immunol Res. 2019;7(1):100–12. doi: 10.1158/2326-6066.CIR-18-0307. DOI: https://doi.org/10.1158/2326-6066.CIR-18-0307
  27. Barber A, Meehan KR, Sentman CL. Treatment of multiple myeloma with adoptively transferred chimeric NKG2D receptor-expressing T cells. Gene Ther. 2011;18(5):509–16. doi: 10.1038/gt.2010.174. DOI: https://doi.org/10.1038/gt.2010.174
  28. Roybal KT, Rupp LJ, Morsut L, et al. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell. 2016;164(4):770–9. doi: 10.1016/j.cell.2016.01.011. DOI: https://doi.org/10.1016/j.cell.2016.01.011
  29. Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer. 2004;4(6):437–47. doi: 10.1038/nrc1367. DOI: https://doi.org/10.1038/nrc1367
  30. Kosti P, Larios-Martinez KI, Maher J, Arnold JN. Generation of hypoxia-sensing chimeric antigen receptor T cells. STAR Protoc. 2021;2(3):100723. doi: 10.1016/j.xpro.2021.100723. DOI: https://doi.org/10.1016/j.xpro.2021.100723

Keywords:

CD87, uPAR, CAR-T lymphocytes, acute myeloid leukemias

License

Copyright (c) 2022 Clinical Oncohematology

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Downloads

Download data is not yet available.

Downloads

Published

01.10.2022

Issue

Vol. 15 No. 4 (2022)
EXPERIMENTAL STUDIES

How to Cite

Neklesova M.V., Smirnov S.V., Shatilova A.A., Levchuk K.A., Ershova A.E., Silonov S.A. Production of CD87 Antigen-Specific CAR-T Lymphocytes and Assessment of Their In Vitro Functional Activity. Clinical Oncohematology. Basic Research and Clinical Practice. 2022;15(4):340–348. doi:10.21320/2500-2139-2022-15-4-340-348.

Most read articles by the same author(s)