The CARTEL^TM AND gate is a genetic circuit which we have designed to be integrated in T cells, for a controlled expression of one output, the chimeric antigen receptor, in response to two inputs that are only found in a tumor microenvironment. The AND gate is constructed of two promoters interconnected through a protein, (HIF1A), to integrate two inputs into one output. Two alternative AND Gates were designed as follows: high VEGF concentrations and hypoxic conditions or alternatively low pH and hypoxic conditions.

As a proof of concept we characterized all the promoters separately in mammalian cell lines. We therefore generated stable cell lines, containing the promoters for the inputs driving a reporter protein. Single and multiple enhancer elements for the inputs: pH, VEGF and hypoxia were designed and integrated in Jurkat and HEK293T cells.
. We could show an induced induced activity of the hypoxia response element promoter and the cAMP response element promoter in Jurkat cells (Fig. 1) Flow cytometry of Jurkat 4xHRE-pTal:eCFP cells showed an increase of eCFP positive cells with rising concentration of CoCl2 (Fig. 1a). In addition we could show an induced activity of the cAMP response element promoter in Jurkat 4xCRE-pTal:eCFP cells treated with forskolin and IBMX (Fig. 1b). With this results, we can demonstrate the our construct 4xHRE-pTal:eCFP and 4xCRE-pTal:eCFP are functional and responsive to hypoxic conditions and low pH, respectively.

The CARTEL^TM AND gate requires the of the endogenous gene coding for hypoxia-inducible factor 1 alpha (HIF1A) to ensure exclusive control over HIF1A by the introduced AND gate. We generated stable Jurkat and HEK293T cell lines in which HIF1A was efficiently knock down. .In addition we could show that transient expression of HA tagged HIF1A in knockdown cells behaves as the endogenous HIF1A, without being affected by the siRNA used to generate the (Fig. 2).

As an additional safety feature for the CARTEL^{TM T cells we generated stable Jurkat T cell lines with the} gene thymidine kinase. In the event of uncontrolled actions by the T cells, they can be eliminated by the administration of the drug ganciclovir. It is only affecting cells containing the kill switch, unmodified cells remain intact. To evaluate the efficiency of our kill switch we performed an apoptosis assay with ganciclovir (Fig. 3). Both cell lines expressing HSV-TK show significant reduced survival rate compared to wild type cells following treatment with ganciclovir. We were able to show that we can add an additional level of safety to our project design by introducing a functional kill switch.

This summer we could demonstrate that all parts of the CARTEL^TM AND gate are separately functional. After optimization and improvement of individual parts the system needs to be tested in primary T cells and may then proceed to pre-clinical studies with animal models. To learn more about possible improvements and future applications visit our