The Ambrosia consists of several parts which carries different aspects of our design, including killing component to clear the senescent cells and control element to regulate the activation and shutdown of CAR-T.

Firstly, the clearance of senescent cells is achieved by the engineered chimeric antigen receptors (CAR)-T cells, which specifically target the senescent marker, Dipeptidyl Peptidase 4 (DPP4). Previous studies reveal that DPP4 was selectively expressed on the surface of senescent (Kim et al., 2017), but not proliferating, human diploid fibroblasts, which suggests the elimination of DPP4 highly expressed cells can delay fibrosis and reverse senescence.

Besides, we were concerned about the safety of our product, so we made some designs to control the activation and shutdown of CAR-T cells. We use caffeine-operated synthetic module (COSMO) to control the switch-on of CAR-T. Several negative feedback loops based on the environmental Interlukin-6 (IL-6) were designed to achieve automated stable reversible switch-off of CAR-T to avoid the happening of inflammatory cytokine release syndrome (CRS).

CAR-T is a developing immunotherapy for the treatment of several cancers. This investigational treatment is a sort of gene therapy that reroutes T lymphocytes to eliminate malignant cells (Huang et al., 2020). Its special structure, 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).

CRS is the most common side effects of CAR-T immunotherapy, which occurs when immune cells release too many cytokines into the environment. This will cause the damage of human body. Previous studies have shown that IL-6 is the main cause of CRS (Frey and Porter, 2019). Therefore, we design two switches to control the activation and shutdown of CAR-T cells.

The first one is caffeine-operated synthetic module (COSMO). COSMO enables chemogenetic manipulation of biological processes by caffeine and it was used in our design as a robust chemically induced dimerization system to control the activation of CAR-T cells (Wang et al., 2020). It 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.

Several negative feedback loops based on the environmental concentration of IL-6 was designed to regulate the shutdown of CAR-T cells.

Several negative feedback loops based on the environmental concentration of IL-6 was designed to regulate the shutdown of CAR-T cells. 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 high affinity IL-6 receptor binding domain (FIG. 3).

Now, we have offered a complete set of designs to develop our anti-DPP4 CAR T immunotherapy to reverse the senescent phenotype. We have made it to realize some meaningful improvements of our CAR-T cell including the inducible caffeine switches and IL-6 based negative feedback loops, which achieves the controllable switches of CAR-T cells and reduces the happening of CRS.

Therefore, our senolytic agent – Ambrosia T cell is now created (FIG. 5). After integrated and transformed the required parts (anti-DPP4 CAR, COSMO, and IL-6 based feedback loops) into the T lymphocytes, an engineered CAR-T cell targets to senescent cells was born.