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

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.

• Ekaterina Konstantinovna Zaikova VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341; Institute of Cytology, 4 Tikhoretskii pr-t, Saint Petersburg, Russian Federation, 194064 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341; Институт цитологии РАН, Тихорецкий пр-т, д. 4, Санкт-Петербург, Российская Федерация, 194064
• K.A. Levchuk VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• D.Yu. Pozdnyakov VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• A.A. Daks Institute of Cytology, 4 Tikhoretskii pr-t, Saint Petersburg, Russian Federation, 194064 ; Институт цитологии РАН, Тихорецкий пр-т, д. 4, Санкт-Петербург, Российская Федерация, 194064
• A.Yu. Zaritskey VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• A.V. Petukhov VA Almazov National Medical Research Center, 2 Akkuratova str., Saint Petersburg, Russian Federation, 197341; Institute of Cytology, 4 Tikhoretskii pr-t, Saint Petersburg, Russian Federation, 194064; Sirius University of Science and Technology, 1 Olimpiiskii pr-t, Sochi, Russian Federation, 354340 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341; Институт цитологии РАН, Тихорецкий пр-т, д. 4, Санкт-Петербург, Российская Федерация, 194064; Научно-технологический университет «Сириус», Олимпийский пр-т, д. 1, Сочи, Российская Федерация, 354340

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