Three-dimensional cell culture of chimeric antigen receptor T cells originated from peripheral blood mononuclear cells towards cellular therapies.

BACKGROUND AIMS: With the objective of improving the ex vivo production of therapeutic chimeric antigen receptor (CAR) T cells, we explored the addition of three-dimensional (3D) polystyrene scaffolds to standard suspension cell cultures. METHODS: We aimed to mimic the structural support given by the lymph nodes during in vivo lymphocyte expansion. RESULTS: We observed an increase in cell proliferation compared with standard suspension systems as well as an enhanced cytotoxicity toward cancer cells. Moreover, we directly obtained the CAR T cells from peripheral blood mononuclear cells, thus minimizing the ex vivo manipulation of the therapeutic cells and opening the way to synergies among different cell populations. CONCLUSIONS: We propose the use of commercially available 3D polystyrene systems to improve the current immune cell cultures and resulting cell products for emerging cellular (immuno)therapies.

The target antigen determines the mechanism of acquired resistance to T cell-based therapies.

Despite the revolution of immunotherapy in cancer treatment, patients eventually progress due to the emergence of resistance. In this scenario, the selection of the tumor antigen can be decisive in the success of the clinical response. T cell bispecific antibodies (TCBs) are engineered molecules that include binding sites to the T cell receptor and to a tumor antigen. Using gastric CEA+/HER2+ MKN45 cells and TCBs directed against CEA or HER2, we show that the mechanism of resistance to a TCB is dependent on the tumor antigen. Acquired resistant models to a high-affinity-CEA-targeted TCB exhibit a reduction of CEA levels due to transcriptional silencing, which is reversible upon 5-AZA treatment. In contrast, a HER2-TCB resistant model maintains HER2 levels and exhibit a disruption of the interferon-gamma signaling. These results will help in the design of combinatorial strategies to increase the efficacy of cancer immunotherapies and to anticipate and overcome resistances.

Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation.

Immunotherapy has raised high expectations in the treatment of virtually every cancer. Many current efforts are focused on ensuring the efficient delivery of active cytotoxic cells to tumors. It is assumed that, once these active cytotoxic cells are correctly engaged to cancer cells, they will unfailingly eliminate the latter, provided that inhibitory factors are in check. T cell bispecific antibodies (TCBs) and chimeric antigen receptors (CARs) offer an opportunity to test this assumption. Using TCB and CARs directed against HER2, here we show that disruption of interferon-gamma signaling confers resistance to killing by active T lymphocytes. The kinase JAK2, which transduces the signal initiated by interferon-gamma, is a component repeatedly disrupted in several independently generated resistant models. Our results unveil a seemingly widespread strategy used by cancer cells to resist clearance by redirected lymphocytes. In addition, they open the possibility that long-term inhibition of interferon-gamma signaling may impair the elimination phase of immunoediting and, thus, promote tumor progression.