Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor

Aim. To study antitumor cytotoxic effect of CAR-T NKG2D and CAR-T anti-CD19 in vitro and in vivo in order to compare antitumor activity of chimeric antigen receptors (CAR) with different structural and functional properties.

Materials & Methods. CAR constructions were produced by molecular cloning. CAR-T cell populations were obtained by transduction of healthy donor T-lymphocytes with recombinant lentiviral particles coding CAR NKG2D or CD19 target antigen CAR sequences. CAR-T cell proportion was assessed by FusionRed fluorescence and EGFR membrane receptor imaging. Specific in vitro cytotoxic activity of CAR-T effector cells was analyzed by Real-Time Cytotoxicity Assay (RTCA) during co-cultivation with HeLa_CD19 target cell line using xCELLigence. Interferon-γ (IFN-γ) synthesis in vitro and in vivo along with the degree of cytotoxic effect were analyzed by immunoassay of culture medium of co-cultivated effector cells and target cells as well as isolated auto-plasma from the peripheral blood of mice. To assess the in vivo functional activity, CAR-T cell populations were infused into immunodeficient NSG-SGM3 mice (10 000 000 cells/mouse) 12 days after HeLa_CD19 cell injection and confirmation of engraftment and tumor growth. Upon euthanasia, tumors were removed and fixed in paraffin to prepare histological sections. CAR-T cell tumor infiltration was assessed by CD3 antigen immunohistochemical staining.

Results. The highest ligand (molecules MICA, ULBP1/2/3/4/5/6) expression levels were detected in HeLa cell line. The obtained NKG2D CAR-T cells showed a considerable cytotoxic activity against HeLa_CD19 target line (cell index [CI] = 1.27), which was, however, twice as low as that of CAR-T anti-CD19 (CI = 0.60) (p = 0.0038). IFN-γ level during co-cultivation of CAR-T anti-CD19 with HeLa_CD19 at the ratio of Е/Т = 1:1 was 64,852 pcg/mL, which was 3.5 times higher than IFN-γ level during co-cultivation of CAR-T NKG2D with HeLa_CD19 (18,635 pcg/mL) (p = 0.0360). The degree of tumor infiltration by CAR-T anti-CD19 cells was higher than that by CAR-T NKG2D. The absence of NKG2D proliferating CAR-T cells in mice peripheral blood confirms their low persistence. IFN-γ concentration in mice auto-plasma was 11.89 pcg/mL after CAR-T anti-CD19 infusion and 0.57 pcg/mL after CAR-T NKG2D infusion (p = 0.0079). The mean weight of tumor xenografts in experimental groups 10 days after CAR-T anti-CD19 injection was 0.72 g (p = 0.0142), after Т-lymphocyte and NKG2D CAR-T cell infusions it was 2.12 g and 1.2 g, respectively.

Conclusion. CAR-T anti-CD19 cells are characterized by more pronounced cytotoxic effect under both in vitro and in vivo experimental conditions compared with CAR-T NKG2D cells. The degree of CAR-T anti-CD19 proliferation and their infiltration in mice xenograft models is considerably higher than the levels reached with NKG2D CAR-T cell injections. A single CAR-T NKG2D injection results only in short-term tumor reduction.

• Kseniya Aleksandrovna Levchuk VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• S.A. Osipova VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• A.V. Onopchenko VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• M.L. Vasyutina VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341
• E.R. Bulatov Kazan (Privolzhsky) Federal University, 18 Kremlevskaya ul., Kazan, Russian Federation, 420008 ; ФГАОУ ВО «Казанский (Приволжский) федеральный университет», ул. Кремлевская, д. 18, Казань, Российская Федерация, 420008
• A.Kh. Valiullina Kazan (Privolzhsky) Federal University, 18 Kremlevskaya ul., Kazan, Russian Federation, 420008 ; ФГАОУ ВО «Казанский (Приволжский) федеральный университет», ул. Кремлевская, д. 18, Казань, Российская Федерация, 420008
• O.N. Demidov VA Almazov National Medical Research Center, 2 Akkuratova ul., 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; ФГБУН «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
• A.V. Petukhov VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341 ; ФГБУ «НМИЦ им. В.А. Алмазова» Минздрава России, ул. Аккуратова, д. 2, Санкт-Петербург, Российская Федерация, 197341

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