CD19

Efficient Transduction of T-Lymphocytes by Lentiviral Particles in Oncoimmunological Studies

AUTOIMMUNE THROMBOCYTOPENIA

EK Zaikova1,2, KA Levchuk1, DYu Pozdnyakov1, AA Daks2, AYu Zaritskey1, AV Petukhov1,2,3

1 VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341

2 Institute of Cytology, 4 Tikhoretskii pr-t, Saint Petersburg, Russian Federation, 194064

3 Sirius University of Science and Technology, 1 Olimpiiskii pr-t, Sochi, Russian Federation, 354340

For correspondence: Ekaterina Konstantinovna Zaikova, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341; e-mail: Catherine3452@yandex.ru

For citation: Zaikova EK, Levchuk KA, Pozdnyakov DYu, et al. Efficient Transduction of T-Lymphocytes by Lentiviral Particles in Oncoimmunological Studies. Clinical oncohematology. 2020;13(3):295–306 (In Russ).

DOI: 10.21320/2500-2139-2020-13-3-295-306

ABSTRACT

Aim. To compare different methods of lentivirus concentration in order to select the best way of providing high-level transduction for generating laboratory CAR-T cells.

Materials & Methods. Concentration of lentiviral supernatant was carried out by 4 methods: ultrafiltration, ultracentrifugation, polyethylene glycol (PEG), water-soluble non-ionic polymer, precipitation method, and ion-exchange chromatography. Functional viral titer was determined by mCherry reporter protein expression in the transduced HeLa cell line as well as by rapid immunochromatographic (IC) tests. Physical titer was determined by ELISA. Transduction efficiency of healthy donor’s T-lymphocytes was assessed by flow cytometry with respect to signal intensity of reporter protein FusionRed. Functional activity of generated anti-CD19 CAR-T was evaluated by microscopy after co-cultivation with CD19-HeLa+ cell line as well as subsequent cytokine testing.

Results. Lentivirus purification and concentration by ultrafiltration provided the greatest number of transduced cells, i.e. 84.7 %. Methods of ultracentrifugation, PEG precipitation, and ion-exchange chromatography yielded 56.08 %, 74.22 %, and 21.05 % of T-cell transduction, respectively. Results of rapid IC tests were comparable (r = 0.91) with cell line titer data. The mean T-cell transduction efficiency was 59.55 % ± 2.94 %, and its maximum reached 76.26 %.

Conclusion. The focus was laid on optimization of CAR-T cell production during the generation of lentiviral vectors and their purification. Ultrafiltration was selected as the best method of lentiviral supernatant concentration to efficiently transduce T-lymphocytes and to generate functional CAR-T cell population.

Keywords: CAR-T lymphocytes, CD19, recombinant lentivirus, lentivirus concentration.

Received: April 29, 2020

Accepted: June 25, 2020

Read in PDF

REFERENCES

  1. Blaese R, Culver K, Miller A, et al. T Lymphocyte-Directed Gene Therapy for ADA-SCID: Initial Trial Results After 4 Years. Science. 1995;270(5235):475–80. doi: 10.1126/science.270.5235.475.

  2. Kochenderfer J, Feldman S, Zhao Y, et al. Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor. J Immunother. 2009;32(7):689–702. doi: 10.1097/cji.0b013e3181ac6138.

  3. Brentjens R, Latouche J, Santos E, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nat Med. 2003;9(3):279–86. doi: 10.1038/nm827.

  4. Pule M, Savoldo B, Myers G, et al. Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma. Nat Med. 2008;14(11):1264–70. doi: 10.1038/nm.1882.

  5. Kochenderfer J, Dudley M, Feldman S, 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):2709–20. doi: 10.1182/blood-2011-10-384388.

  6. Brentjens R, Davila M, 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. doi: 10.1126/scitranslmed.3005930.

  7. Lewinski M, Bushman F. Retroviral DNA Integration—Mechanism and Consequences. Adv Genet. 2005;55:147–81. doi: 10.1016/s0065-2660(05)55005-3.

  8. Kochenderfer J, Dudley M, Carpenter R, et al. Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation. Blood. 2013;122(25):4129–39. doi: 10.1182/blood-2013-08-519413.

  9. Cruz C, Micklethwaite K, Savoldo B, et al. Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study. Blood. 2013;122(17):2965–73. doi: 10.1182/blood-2013-06-506741.

  10. Grupp S, Kalos M, Barrett D, et al. Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia. N Engl J Med. 2013;368(16):1509–18. doi: 10.1056/nejmoa1215134.

  11. Kalos M, Levine B, Porter D, 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. doi: 10.1126/scitranslmed.3002842.

  12. Qin D, Huang Y, Li D, et al. Paralleled comparison of vectors for the generation of CAR-T cells. Anticancer Drugs. 2016;27(8):711–22. doi: 10.1097/cad.0000000000000387.

  13. Gogol-Doring A, Ammar I, Gupta S, et al. Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4+ T Cells. Mol Ther. 2016;24(3):592–606. doi: 10.1038/mt.2016.11.

  14. Izsvak Z, Hackett P, Cooper L, Ivics Z. Translating Sleeping Beauty transposition into cellular therapies: Victories and challenges. BioEssays. 2011;33(6):478–9. doi: 10.1002/bies.201190025.

  15. Manuri P, Wilson M, Maiti S, et al. piggyBac Transposon/Transposase System to Generate CD19-Specific T Cells for the Treatment of B-Lineage Malignancies. Hum Gene Ther. 2010;21(4):427–37. doi: 10.1089/hum.2009.114.

  16. Nakazawa Y, Huye L, Salsman V, et al. PiggyBac-mediated Cancer Immunotherapy Using EBV-specific Cytotoxic T-cells Expressing HER2-specific Chimeric Antigen Receptor. Mol Ther. 2011;19(12):2133–43. doi: 10.1038/mt.2011.131.

  17. Barrett D, Zhao Y, Liu X, et al. Treatment of Advanced Leukemia in Mice with mRNA Engineered T Cells. Hum Gene Ther. 2011;22(12):1575–86. doi: 10.1089/hum.2011.070.

  18. Dai X, Park J, Du Y, et al. One-step generation of modular CAR-T cells with AAV–Cpf1. Nat Meth. 2019;16(3):247–54. doi: 10.1038/s41592-019-0329-7.

  19. Mann R, Mulligan R, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983;33(1):153–9. doi: 10.1016/0092-8674(83)90344-6.

  20. Le Doux J, Davis H, Morgan J, Yarmush M. Kinetics of retrovirus production and decay. Biotechnol Bioeng. 1999;63(6):654–62. doi: 10.1002/(sici)1097-0290(19990620)63:6<654::aid-bit3>3.0.co;2-1.

  21. Andreadis S, Brott D, Fuller A, Palsson B. Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours. J Virol. 1997;71(10):7541–8. doi: 10.1128/jvi.71.10.7541-7548.1997.

  22. Naldini L, Blomer U, Gallay P, et al. In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector. Science. 1996;272(5259):263–7. doi: 10.1126/science.272.5259.263.

  23. Sakuma T, Barry M, Ikeda Y. Lentiviral vectors: basic to translational. Biochem J. 2012;443(3):603–18. doi: 10.1042/bj20120146.

  24. Dull T, Zufferey R, Kelly M, et al. A Third-Generation Lentivirus Vector with a Conditional Packaging System. J Virol. 1998;72(11):8463–71. doi: 10.1128/jvi.72.11.8463-8471.1998.

  25. Vannucci L, Lai M, Chiuppesi F, et al. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol. 2013;36(1):1–22.

  26. Modlich U, Navarro S, Zychlinski D, et al. Insertional Transformation of Hematopoietic Cells by Self-inactivating Lentiviral and Gammaretroviral Vectors. Mol Ther. 2009;17(11):1919–28. doi: 10.1038/mt.2009.179.

  27. Sastry L, Xu Y, Duffy L, et al. Product-Enhanced Reverse Transcriptase Assay for Replication-Competent Retrovirus and Lentivirus Detection. Hum Gene Ther. 2005;16(10):1227–36. doi: 10.1089/hum.2005.16.1227.

  28. Cornetta K, Yao J, Jasti A, et al. Replication-competent Lentivirus Analysis of Clinical Grade Vector Products. Mol Ther. 2011;19(3):557–66. doi: 10.1038/mt.2010.278.

  29. Sena-Esteves M, Gao G. Titration of Lentivirus Vectors. Cold Spring Harb Protoc. 2018;2018(4):pdb.prot095695. doi: 10.1101/pdb.prot095695.

  30. Kutner R, Zhang X, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc. 2009;4(4):495–505. doi: 10.1038/nprot.2009.22.

  31. Merten O, Hebben M, Bovolenta C. Production of lentiviral vectors. Mol Ther Meth Clin Devel. 2016;3:16017. doi: 10.1038/mtm.2016.17.

  32. Sena-Esteves M, Tebbets J, Steffens S, et al. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Meth. 2004;122(2):131–9. doi: 10.1016/j.jviromet.2004.08.017.

  33. Boudeffa D, Fenard D, Mormin M, et al. Development of Innovative Scalable Protocols for the Purification of Lentiviral Vectors Pseudotyped With GaLV-TR or Mutated Measles Virus Glycoproteins. Mol Ther. 2015;23:S214–S215. doi: 10.1016/s1525-0016(16)34143-0.

  34. Baekelandt V, Eggermont K, Michiels M, et al. Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain. Gene Ther. 2003;10(23):1933–40. doi: 10.1038/sj.gt.3302094.

  35. Sanber K, Knight S, Stephen S, et al. Construction of stable packaging cell lines for clinical lentiviral vector production. Sci Rep. 2015;5(1):9021. doi: 10.1038/srep09021.

  36. Зайцев Д.В., Зайкова Е.К., Головкин А.С. и др. Гранулоцитарно-макрофагальный колониестимулирующий фактор и технология CAR-T при солидных опухолях в эксперименте. Клиническая онкогематология. 2020;13(2):115–22. doi: 10.21320/2500-2139-2020-13-2-115-122.[Zaytsev DV, Zaikova EK, Golovkin AS, et al. Granulocyte-Macrophage Colony-Stimulating Factor and CAR-T Technology for Solid Tumors in Experiment. Clinical oncohematology. 2020;13(2):115–22. doi: 10.21320/2500-2139-2020-13-2-115-122. (In Russ)]

  37. Петухов, А.В., Маркова, В.А., Моторин, Д.В. и др. Получение CAR T-лимфоцитов, специфичных к CD19, и оценка их функциональной активности in vitro. Клиническая онкогематология. 2018;11(1):1–9. doi: 10.21320/2500-2139-2018-11-1-1-9.[Petukhov AV, Markova VA, Motorin DV, et al. Manufacturing of CD19 Specific CAR T-Cells and Evaluation of their Functional Activity in Vitro. Clinical oncohematology. 2018;11(1):1–9. doi: 10.21320/2500-2139-2018-11-1-1-9. (In Russ)]

Clinical Significance of Immunophenotyping of Bone Marrow Cells in Multiple Myeloma

LYMPHOID TUMORS , , , , ,

OYu Yakimovich, OM Votyakova, NV Lyubimova, NN Tupitsyn

N.N. Blokhin Russian Cancer Research Center, 24 Kashirskoye sh., Moscow, Russian Federation, 115478

For correspondence: Oksana Yur’evna Yakimovich, graduate student, 24 Kashirskoye sh., Moscow, Russian Federation, 115478; Tel.: +7(499)324-28-54; e-mail: ronc_ramn@mail.ru

For citation: Yakimovich OYu, Votyakova OM, Lyubimova NV, Tupitsyn NN. Clinical Significance of Immunophenotyping of Bone Marrow Cells in Multiple Myeloma. Clinical oncohematology. 2016;9(3):296-301 (In Russ).

DOI: 10.21320/2500-2139-2016-9-3-296-301

ABSTRACT

Aim. To analyze the relationship between expression of aberrant CD45, CD19, CD56 markers on the plasma cells and clinical and laboratory findings and prognostically significant parameters in patients with multiple myeloma (MM).

Methods. This scientific research includes data on clinical investigation and immunophenotyping of bone marrow cells obtained from 64 MM patients treated in the N.N. Blokhin Russian Cancer Research Center over the period from 2004 to 2015. The three-color flow cytometry was performed using a direct immunofluorescence technique (CD38-PerCP, CD138-FITC monoclonal antibodies) and PE-conjugated monoclonal antibodies against CD45, CD19, and CD56.

Results. Comparison of average values of the total count of plasma cells, the number of plasmablasts, proplasmacyte and mature plasma cells (according to the myelogram) and comparison of these data with the level of expression of the CD19 marker demonstrated a significant relationship between the CD19 negative immunophenotype and both a higher level of the total count of plasma cells and immature plasma cells. There also was a significant correlation between the CD19 negative immunophenotype and a higher level of C-reactive protein, which is significant prognostic factor in MM. In addition, there was a significant relationship between the CD19 negative phenotype and a higher percentage of young neutrophils in blood, i.e. with a more frequent “left shift”. The CD56 negative phenotype is associated with plasmablastic morphology of plasma cells and with the presence of plasma cells in the peripheral blood. Plasma cell leukemia is more common in patients with СD56 negative phenotype of myeloma cells. The CD45 negative immunophenotype was associated with a higher level of k-type FLCs, Bence-Jones proteinuria and with a higher serum creatinine, than in the cases of CD45 positive phenotype.

Conclusion. The study of the immunophenotype of plasma cells in MM has important scientific and practical significance and requires further study.

Keywords: multiple myeloma, aberrant immunophenotype of malignant plasma cells, CD45, CD19, and CD56 markers, clinical and laboratory parameters.

Received: March 17, 2016

Accepted: April 1, 2016

Read in PDF (RUS) pdficon

REFERENCES

  1. Зуева Е.Е., Русанова Е.Б., Куртова А.В. Диагностика множественной миеломы и мониторинг эффективности терапии. Иммунология гемопоэза. 2008;5(2):44–56.
    [Zueva EE, Rusanova EB, Kurtova AV. Diagnosis of multiple myeloma and treatment efficacy monitoring. Immunologiya gemopoeza. 2008;5(2):44–56. (In Russ)]
  2. Bataille R, Jero G, Robillard N, et al. The phenotype of normal, reactive and malignant plasma cells. Identification of “many and multiple myelomas” and of new targets for myeloma therapy. Haematologica. 2006;91(9):1234–40.
  3. de Tute RM, Jack AS, Child JA, et al. A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma. Leukemia. 2007;21(9):2046–9. doi: 10.1038/sj.leu.2404815.
  4. Johnsen HE, Bogsted М, Klausen TW, et al. Multiparametric flow cytometry profiling of neoplastic plasma cells in multiple myeloma. Cytometry B Clin Cytom. 2010;78В(5):338–47. doi: 10.1002/cyto.b.20523.
  5. Manzanera GM, San Miguel Izquierdo JF, de Matos OA. Immunophenotyping of plasma cells in multiple myeloma. Meth Mol Med. 2005;113:5–24. doi: 10.1385/1-59259-916-8:5.
  6. Mateo G, Castellanos M, Rasillo A, et al. Genetic abnormalities and patterns of antigenic expression in multiple myeloma. Clin Cancer Res. 2005;11(10):3661–7. doi: 10.1158/1078-0432.CCR-04-1489.
  7. Ocqueteau M, Orfao A, Almeida J, et al. Immunophenotypic characterization of plasma cells from monoclonal gammopathy of undetermined significance patients. Implications for the differential diagnosis between MGUS and multiple myeloma. Am J Pathol. 1998;152(6):1655–65.
  8. Perez-Persona E, Vidriales MB, Mateo G, et al. New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smouldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells. Blood. 2007;110(7):2586–92. doi: 10.1182/blood-2007-05-088443.
  9. Rawstron AC, Davies FE, Das Gupta R, et al. Flow cytometric disease monitoring in multiple myeloma: The relationship between normal and neoplastic plasma cells predicts outcome after transplantation. Blood. 2002;100(9):3095–100. doi: 10.1182/blood-2001-12-0297.
  10. Rawstron AC, Orfao A, Beksac M, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica. 2008;93(3):431–8. doi: 10.3324/haematol.11080.
  11. Robillard N, Pellat-Deceunynck C, Bataille R. Phenotypic characterization of the human myeloma cell growth fraction. Blood. 2005;105(12):4845–8. doi: 10.1182/blood-2004-12-4700.
  12. San Miguel JF, Almeida J, Mateo G, et al. Immunophenotypic evaluation of the plasma cell compartment in multiple myeloma: A tool for comparing the efficacy of different treatment strategies and predicting outcome. Blood. 2002;99(5):1853–6. doi: 10.1182/blood.v99.5.1853.
  13. Sezer O, Heider U, Zavrski I, Possinger K. Differentiation of monoclonal gammopathy of undetermined significance and multiple myeloma using flow cytometric characteristics of plasma cells. Haematologica. 2001;86(8):837–43.
  14. Moreau P, Robillard N, Avet-Loiseau H, et al. Patients with CD45 negative multiple myeloma receiving high-dose therapy have a shorter survival than those with CD45 positive multiple myeloma. Haematologica. 2004;89(5):547–51.
  15. Pellat-Deceunynck C, Barille S, Jego G, et al. The absence of CD56 (NCAM) on malignant plasma cells is a hallmark of plasma cell leukemia and of a special subset of multiple myeloma. Leukemia. 1998;12(12):1977–82. doi: 10.1038/sj.leu.2401211.
  16. Jego G, Avet-Loiseau H, Robillard N, et al. Reactive plasmacytoses in multiple myeloma during hematopoietic recovery with G- or GM-CSF. Leuk Res. 2000;24(7):627–30. doi: 10.1016/s0145-2126(00)00033-3.
  17. Sarasquete ME, Garcia-Sanz R, Gonzalez D, et al. Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry. Haematologica. 2005;90(10):1365–72.
  18. Ishikawa H, Tsuyama N, Abroun S, et al. Requirements of src family kinase activity associated with CD45 for myeloma cell proliferation by interleukin-6. Blood. 2002;99(6):2172–8. doi: 10.1182/blood.v99.6.2172.
  19. Robillard N, Wuilleme S, Lode L, et al. CD33 is expressed on plasma cells of a significant number of myeloma patients, and may represent a therapeutic target. Leukemia. 2005;19(11):2021–2. doi: 10.1038/sj.leu.2403948.
  20. Tassone P, Goldmacher VS, Neri P, et al. Cytotoxic activity of the maytansinoid immunoconjugate B-B4- DM1 against CD138+ multiple myeloma cells. Blood. 2004;104(12):3688–96. doi: 10.1182/blood-2004-03-0963.
  21. Treon SP, Raje N, Anderson KC. Immunotherapeutic strategies for the treatment of plasma cell malignancies. Semin Oncol. 2000;27(5):598–613.
  22. Тупицын Н.Н. Иммунология клеток крови. В кн.: Гематология. Национальное руководство. Под ред. О.А. Рукавицына. М.: ГЭОТАР-Медиа, 2015. С. 70–8.
    [Tupitsyn NN. Blood cell immunology. In: Rukavitsyn OA, ed. Gematologiya. Natsional’noe rukovodstvo. (Hematology. National guidelines.) Moscow: GEOTAR-Media Publ.; 2015. pp. 70–8. (In Russ)]
  23. Менделеева Л.П., Вотякова О.М., Покровская О.С. и др. Национальные клинические рекомендации по диагностике и лечению множественной миеломы. Гематология и трансфузиология. 2014;59(приложение 3):1–37.
    [Mendeleeva LP, Votyakova OM, Pokrovskaya OS, et al. National clinical guidelines for diagnosis and treatment of multiple myeloma. Gematologiya i transfuziologiya. 2014;59(Suppl. 3):1–37. (In Russ)]
  24. Lin Р. Плазмоклеточная миелома. Прогресс в лечении множественной миеломы. Best Clin Pract, русское издание. 2009;2:11–6.
    [Lin P. Plasma cell myeloma. Progress in treatment of multiple myeloma. Best Clin Pract, Russian edition. 2009;2:11–6 (In Russ)]
  25. Вотякова О.М., Любимова Н.В., Турко Т.А. и др. Клиническое значение исследования свободных легких цепей иммуноглобулинов при множественной миеломе. Вестник РОНЦ им. Н.Н. Блохина РАМН. 2010;5(4):16–20.
    [Votyakova OM, Lyubimova NV, Turko TA, et al. Clinical implication of immunoglobulin free light chains study in patients with multiple myeloma. Vestnik RONTs im. N.N. Blokhina RAMN. 2010;5(4):16–20. (In Russ)]