Expansion of human T cells for adoptive immunotherapy using a bisphosphonate prodrug

Cancer immunotherapy with human T cells expressing V2V2 T cell receptor (also termed V9V2) has shown promise because of their ability to recognize and kill most types of tumors in a major histocombatibility complex (MHC) -unrestricted fashion that is independent of the number of tumor mutations. In clinical trials, adoptive transfer of V2V2 T cells has been shown to be safe and does not require preconditioning. In this report, we describe a method for preparing highly enriched human V2V2 T cells using the bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA). PTA stimulated the expansion of V2V2 cells to purities up to 99%. These levels were consistently higher than those observed after expansion with zoledronic acid, the most commonly used stimulator for clinical trials. Cell numbers also averaged more than those obtained with zoledronic acid and the expanded V2V2 cells exhibited high cytotoxicity against tumor cells. The high purity of V2V2 cells expanded by PTA increased engraftment success in immunodeficient NOG mice. Even low levels of contaminating T cells resulted in some mice with circulating human T cells rather than V2V2 cells. V2V2 cells from engrafted NOG mice upregulated CD25 and secreted tumor necrosis factor- and interferon- in response to PTA-treated tumor cells. Thus, PTA expands V2V2 T cells to higher purity than zoledronic acid. The high purities allow the successful engraftment of immunodeficient mice without further purification and may speed up the development of allogeneic V2V2 T cell therapies derived from HLA-matched normal donors for patients with poor autologous V2V2 T cell responses.