Phenotypic and functional characteristics of GD2-specific murine CAR T cells in vitro and in vivo

• Julia G. Philippova
• Yulia A. Shevchenko
• Kirill V. Nazarov
• Vasily V. Kurilin
• Marina S. Fisher
• Hiroshi Shiku
• Sergey V. Sennikov

Abstract

Introduction. The adoptive transfer of ex vivo chimeric antigen receptor (CAR)-modified autologous T cells represents a promising approach to the treatment of hematological malignancies, but the treatment of solid tumors remains a challenge. Disialoganglioside (GD2) is a potential target for CAR T cell therapy of solid tumors, due to the high degree of homology between human and murine antigens. The syngeneic murine model with homologous antigen, as an additional preclinical approach, offers significant potential for the optimization of CAR-T technology, and, in turn, may facilitate the successful clinical trials for solid tumors.

Aim of the study was to evaluate the phenotypic and cytotoxic properties of murine GD2-specific CAR T cells in vitro, and their antigen-specific antitumor activity in vivo in the syngeneic B78-21 melanoma model.

Material and methods. The study was conducted using mice of the C57Bl/6 line. Murine CD3⁺ T cells isolated by magnetic separation were transduced with a retroviral vector enco­ding the GITR ligand (GITRL) and a GD2-specific CAR. The phenotype of GD2-specific CAR T cells was analyzed by flow cytometry with regard to the T cell memory markers CD44 and CD62L, and the T cell depletion markers PD-1 and TIM-3. The in vitro cytotoxic characteristics of transduced cells co-cultivated with target cells (GD2⁺ cells B78-21 and GD2^– cells B78-P4) were evaluated by the assay of lactate dehydrogenase content in conditioned media and by flow cytometry to determine the presence of activation markers (CD69, CD137, CD154) and cytotoxicity markers (CD107a, CD178). The in vivo antitumor activity of the transduced cells was investigated using the GD2-positive syngeneic B78-21 melanoma model and a single administration of GD2-specific CAR T cells at a dose of 5 - 10⁶ cells per mouse.

Results. The generation of murine CAR T cells through retroviral transduction resulted in the production of the population of CD8⁺ cells that was twice the size of the CD4⁺ cells proportion. This population is dominated by effector memory CD44⁺CD62L^– cells, but transduction resulted in an increase in the proportion of central memory CD44⁺CD62L⁺ cells and cells expressing the TIM-3 exhaustion marker in both population. The interaction with GD2⁺ target cells resulted in a significant increase in the proportion of transduced cells expressing the activation markers CD69, CD137, CD154, the cytotoxicity marker CD107a, and a discernible tendency for an increase in the marker CD178. The in vitro cytotoxicity assay demonstrated that the transduced T cells not only exhibited cytotoxic potential, but also demonstrated antigen specificity for GD2⁺ tumor cells. The in vivo antitumor activity of murine CAR T cells was demonstrated by their capacity to inhibit tumor node growth, and the survival rate of tumor-bearing mice was 85 days. The median overall survival of mice following infusion of transduced cells was 61,5 days, which is twice as long as the control groups.

Conclusion. The resulting murine CAR T cells have the phenotype of cytotoxic T lymphocytes and exhibit antigen-specific properties both in vitro, against GD2-positive tumor cell lines, and in vivo, in the syngeneic B78-21 melanoma model.

Keywords: GD2; murine CAR T cells; retroviral transduction; cytotoxicity; syngeneic B78-21 melanoma model

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