Background CAR/T Cell therapy relies on Cells with engineeredreceptors that bind tumour cells, then transduces a stimulatory signal meant toactivate the engineered Cell to kill the tumour cell. Clinical experience hasindicated that activating T-cells through its TCR alone isn’t going to besufficient to mount and maintain an effective anti-tumour response. Addingadditional strength to the T cells by providing a 2nd or 3rd engineeredco-stimulatory signal are currently pursued strategies within Celltherapy. In vitro assays are needed to evaluate the effect of theadded co-stimulatory signals and to evaluate the potency of engineered T cells.Most current assays are based on cell-cell interaction which makes them hard tostandardize. We have exploredDextramer technology as an artificial antigen-presenting scaffolds able tostimulate and activate specific T cells. Dextramer displaying MHCp complexesand anti-CD28 antibody (MHC/a-CD28 Dextramer) were used to stimulate PBMCs oran TCR engineered T cell line.

Materials and Methods Healthy donor PBMCs compricing virus-specificCD8+ T cells or TCR engineered T cell lines were incubated with MHC/a-CD28 Dextramer in culture medium for6 hours to 14 days. Antigen specific T cells were following analyzed for i)activation by upregulation of CD69 and CD137, ii) cytokine production (IFN-gamma,TNF-alpha) and iii) proliferation.

Results MHC/a-CD28 Dextramer couldstimulate an activate specific T cells in PBMC sample: i) an upregulation of earlyactivation markers, CD69 and CD137 was observed, ii) increase in cytokineproduction, INF-gamma and TNF-alpha could be measured intracellular, and iii) a15 fold expansion of T cells specific for the MHCp complex of the Dextramer wasmeasured, all proliferating cells were expressing Ki-67.Nostimulation or activation of T cells were observed when incubating cellssamples with MHC/a-CD28 Dextramer displaying negative control MHCp.Similar results wereobtained when stimulating engineered T cell line.

Conclusions We have shown a simple technology to explore effectof engineered co-stimulatory signals, and evaluate potency ofengineered T cells. MHC/a-CD28 Dextramer were able to stimulate, activate andexpand specific T cells. The activation was highly antigen (i.e.MHCp) specific and CD28 co-immobilization dependent.

B. Hansen: A. Employment (full or part-time); Modest; Immudex Aps. J. Jørgensen: A. Employment (full or part-time); Modest; Immudex Aps. L. Brix: A. Employment (full or part-time); Modest; Immudex Aps..