[Frontiers In Bioscience, Landmark, 22, 1644-1654, June 1, 2017]

FLAG-tagged CD19-specific CAR-T cells eliminate CD19-bearing solid tumor cells in vitro and in vivo

Robert Berahovich1, Shirley Xu1, Hua Zhou1, Hizkia Harto1, Qumiao Xu1, Andres Garcia1, Fenyong Liu2, Vita Golubovskaya1, Lijun Wu1

1ProMab Biotechnologies, Richmond, CA, 2Department of Infectious Diseases, University of California, Berkeley, CA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell lines
3.2. CAR constructs
3.3. Generation of CAR-encoding lentivirus
3.4. Generation and expansion of CAR-T cells
3.5. Flow cytometry
3.6. Generation of the stable HeLa-CD19 cell line
3.7. Real-time cytotoxicity assay (RTCA)
3.8. Cytokine induction assay
3.9. Mouse tumor studies
3.10. Immunohistochemistry (IHC)
3.11. Statistical analysis
4. Results
4.1. CD19-FLAG CAR-T cells expand >100 fold in vitro
4.2. CD19-FLAG CAR-T cells exhibit strong CD19-dependent cytolytic activity
4.3. CD19-FLAG CAR-T cells secrete IFN-γ and IL-2 in response to CD19+ but not CD19- tumor cells
4.4. CD19-FLAG CAR-T cells inhibit CD19+ solid tumors in vivo
4 5. CD19-FLAG CAR-T cells inhibit CD19+ hematological cancer in vivo
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Autologous T cells expressing chimeric antigen receptors (CARs) specific for CD19 have demonstrated remarkable efficacy as therapeutics for B cell malignancies. In the present study, we generated FLAG-tagged CD19-specific CAR-T cells (CD19-FLAG) and compared them to their non-tagged counterparts for their effects on solid and hematological cancer cells in vitro and in vivo. For solid tumors, we used HeLa cervical carcinoma cells engineered to overexpress CD19 (HeLa-CD19), and for hematological cancer we used Raji Burkitt's lymphoma cells, which endogenously express CD19. Like non-tagged CD19 CAR-T cells, CD19-FLAG CAR-T cells expanded in culture >100-fold and exhibited potent cytolytic activity against both HeLa-CD19 and Raji cells in vitro. CD19-FLAG CAR-T cells also secreted significantly more IFN-gamma and IL-2 than the control T cells. In vivo, CD19-FLAG CAR-T cells significantly blocked the growth of HeLa-CD19 solid tumors, increased tumor cleaved caspase-3 levels, and expanded systemically. CD19-FLAG CAR-T cells also significantly reduced Raji tumor burden and extended mouse survival. These results demonstrate the strong efficacy of FLAG-tagged CD19 CAR-T cells in solid and hematological cancer models.

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Abbreviations: CAR, Chimeric Antigen Receptor; scFv, Single Chain Variable Fragment, PBS, Phosphate-Buffered Saline

Key Words: Chimeric antigen receptor, immunotherapy, cancer, cytokine, cell

Send correspondence to: Vita Golubovskaya, Promab Biotechnologies, 2600 Hilltop Drive, Richmond, CA 94806, Tel: 510-974-0694, Fax: 510-740-3625, E-mail: vita.gol@promab.com