Our Ambrosia T is divided into two parts, killing elements and control elements. For killing elements, we engineered an anti-Dipeptidyl Peptidase 4 (DPP4) chimeric antigen receptor (CAR), containing anti-DPP4 single chain fragment variable (scFv), CD8 transmembrane domain, 4-1BB co-stimulator domain, and CD3-ζ domain. And for control elements, we incorporated a caffeine-operated synthetic module (COSMO) and three IL-6-based negative feedback loops to control the activation and inactivation of CAR-T cells. In the same way, our experiment consisted of two parts, verification of the lethality of CAR-T cells and verification of the effect of switches, which jointly prove the feasibility of our design. CAR structures and sequences we provided demonstrated superior killing efficiency in the experiments. Moreover, we expressed switches separately with anti-CD19 CAR in Jurkat cells and found that cells equipped with IL-6R-PD-1 showed reduced of cytotoxicity, which proves that our negative feedback element regulation is effective.

CARs are unique receptors that are designed to target a specific antigen to functionally reprogram T lymphocytes. When the CARs recognize to their targets, it will activate and promote the proliferation of T cells and secretion of granzymes and perforins. Then, the target cells will be killed. Since the killing properties of T cells target all target cells, targeted killing of senescent cells by CAR-T cells is possible. DPP4 was a potential senescent marker which selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. We engineered an anti-DPP4 CAR, containing anti-DPP4 scFv, CD8 transmembrane domain, 4-1BB co-stimulator domain, and CD3-ζ domain. When the engineered CAR-T cells recognize the DPP4 highly expressed senescent cells, the downstream 4-1BB and CD3-ζ will be activated to promote the proliferation of T cells and secretion of granzymes and perforins to achieve the goal of killing the senescent cells (FIG. 1).

The COSMO was inserted between 4-1BB co-stimulator domain and CD3-ζ domain. In the absence of caffeine in the environment, when engineered CAR-T cells bind to their respective targets, the CD3- domain cannot be activated due to the blockage of COSMO, and the CAR T cells cannot fully activate, preventing them from carrying out their normal function of killing senescent target cells. However, if the caffeine is presented in the environment, the two engineered CARs can dimerize under the action of COSMO, resulting in the activation of downstream CD3 signaling pathway. Thus, CAR-T cells could be fully activated and perform normal functions (FIG.2). This is a human-controlled CAR-T cell turn-on via COSMO.

Firstly, we designed an anti-IL-6 synthetic Notch receptor (SynNotch). The anti-IL-6 scFv was connected to the Notch regulatory domain which followed by the recombinant transcriptional factor, Gal4-KRAB. When the concentration of IL-6 in the environment is higher than a certain threshold, a large amount of IL6 will bind to anti-IL-6 scFv, leading to the conformational change of SynNotch receptor, which is cleaved by intracellular enzymes. Then the downstream Gal4-KRAB transcription factor is released and transferred to the nucleus, where it binds to the UAS-pSV40 promoter in the nucleus. Consequently, the downstream CAR gene expression was inhibited. The second design was quite similar to the first one, except that the anti-IL-6 scFv was replaced by the low affinity IL-6 receptor binding domain. Moreover, we designed a recombinant receptor which combined the IL-6 receptor extracellular binding domain and PD-1 intracellular domain. When the extracellular IL-6 concentration is high enough, it will bind to the IL-6R extracellular domain and activate the intracellular inhibitory receptors signaling modules. Therefore, the downstream signal of CD3-ζ will be inhibited and inhibit the CAR-T cells activity.