Sleeping Beauty kit sets provide rapid and accessible generation of artificial antigen-presenting cells for natural...

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)-based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80-95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the in vitro expansion of primary cells.