Large-Scale Expansion Of Gamma Delta T Cells And Peptide-Specific Cytotoxic T Cells Using Zoledronate For Adoptive Immunotherapy

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale gamma delta T-cell expansion. To induce expansion of gamma delta T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of gamma delta T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3-specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and 12 pulsed with GPC3 peptide, but not against SK-Hep-l/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including gamma delta T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-l/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of gamma delta T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.