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

AUTOIMMUNE THROMBOCYTOPENIA

KA Levchuk1, SA Osipova1, AV Onopchenko1, ML Vasyutina1, ER Bulatov2, AKh Valiullina2, ON Demidov1,3,4, AV Petukhov1

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

2 Kazan (Privolzhsky) Federal University, 18 Kremlevskaya ul., Kazan, Russian Federation, 420008

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

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

For correspondence: Kseniya Aleksandrovna Levchuk, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341; e-mail: levchuk_ka@almazovcentre.ru

For citation: Levchuk KA, Osipova SA, Onopchenko AV, et al. Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor. Clinical oncohematology. 2022;15(4):327–39. (In Russ).

DOI: 10.21320/2500-2139-2022-15-4-327-339

ABSTRACT

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.

Keywords: CAR-T cell therapy, NKG2D chimeric antigen receptor, co-stimulatory domains, NKG2D ligands, cytotoxic effect, CAR-T infiltration, CAR-T persistence.

Received: June 27, 2022

Accepted: September 12, 2022

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Статистика Plumx английский

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