Page 28 - SaxoCell Annual Report22/23

The protocols were compared in terms of cell yield, CAR expression level, functionality and gene
expression. Subsequently, an SOP for a 7-day manufacturing process was developed and tested
(see figure).

Optimization and shortening of CAR T cell production. T cells are isolated and activated on day 0. On day 2, gene transfer is
performed by sleeping beauty transposition. Cells are then expanded until harvest. Protocols tested included differences
in activators, electroporation devices, and expansion time. The manufacturing process optimized in UltraCAR-T achieves
the desired target dose of CAR-T cells as early as day 7.

The CAR-T cell product was also targeted at the genome and proteome levels, particularly to
determine intrinsic T cell fitness. For this purpose, T cells were examined at the days of T cell
isolation, electroporation, and harvest using nanostring analysis. Nanostring analysis is a
standardized and relatively inexpensive "entry-level" technology that we used to prepare for the
more elaborate analyses on the OMICS platform. It was found that the use of different
manufacturing protocols resulted in single significant differences in the gene expression profile of
the harvested T cells. The changes in gene expression profile were also reflected in protein
expression, which was confirmed using flow cytometric measurements. These results indicate that
manufacturing time in particular influences T cell fitness and differentiation.
As a key result, UltraCART presents a new, innovative CAR-T cell product for the treatment of AML,
and an optimized GMP manufacturing template for CAR-T cells, which will also be used for further
product candidates from T-CURX's pipeline.

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