CD3ζ is a fixed module in all existing chimeric antigen receptor (CAR) as the primary transmitter of endogenous T-cell receptor (Wu et al., 2020). This part is an improved part of cytoplasmic domain of CD3ζ chain (BBa_K3244012) posted by iGEM19_AFCM-Egypt.

Synthetic biology requires the specific control systems that are functionalized to perform user-defined, precisely controlled regulation. Furthermore, considering that anti-aging therapy needs to be continuous, stable, and gentle, choosing common and no-threatening molecules would be more beneficial for public recognition as well as circumventing the adverse effects of drugs.

Using caffeine to modulate CD3ζ signaling is a great potential application in CAR due to its non-toxicity, low cost and easy obtain. COSMO is an optimized caffeine antibody based on wild-type acVHH. It is a robust chemically induced dimerization system with high affinity towards caffeine (EC50 = 16.9 × 10−9 m) (Wang et al., 2021).

It is an improvement over part of NMU_China 2021 design of “Toggle Macrophage: Synthetic Antiviral Macrophages with Immune-regulatory Capacity” (BBa K4040021 and BBa K4040020). This system utilizes IL6R and gp130 to sense IL-6 concentration in the environment (Fig.1①). When low-affinity IL6R binds to IL6 with high concentration, it will recruit gp130, IL-6 cytokine family signal transducer. Its downstream-linked TEV protease will bind to TCS cleavage site, downstream of IL6R, releasing Gal4-KRAB. Eventually, Gal4-KRAB will combine with UAS-pSV40, inhibiting the following gene transcription.

However, due to three long plasmids, it is hard to have high transfection efficiency and difficulty in obtaining enough cells with high positive rates. We hope to simplify the protein structure to shorten the length of plasmids.

Furthermore, cytokine release syndrome (CRS) has been observed in patients with CAR-T treatment, including numerous successful trials, causing severe or even lethal releases of inflammatory cytokine levels, especially IL-6, one of CRS biomarkers (Rafiq et al., 2020). To alleviate CRS, we designed to apply this improved system to inhibit CAR expression by recognizing high concentration of IL-6 to slow down the progress of CRS; and, at the same time, restarts CAR expression at low concentrations to continue killing tumor cells.

We utilized ‘notch’ to replace ‘TCS’ and ‘TEV’, functioning as freeing up Gal4-KRAB as shown in Fig1 ② and ③. The ‘notch’ part is the minimal core transmembrane domain of the wild-type notch, controlling proteolysis (Morsut et al., 2016). With activation of high concentration of IL-6 and sending signaling to the ‘notch’, ‘notch’ will change the conformation and expose multiple proteolytic cleavage site. Various cells have the necessary proteolytic machinery for ‘notch’ activation such that Gal4-KRAB can be released to combine with the UAS-pSV40. Another improved part is that we changed IL6R to IL-6 scFv (Tan Hong Ji and Campana, 2021). ScFv with lower affinity could make sure the negative feedback system only launch when sensing aberrantly high IL-6 levels.

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Blocking expression of CAR-T only can slow down the onset of CRS. If T cell activation can be inhibited rather than blocking new expression of CAR on the cell membrane through high IL-6 level, CRS will be alleviated and relieved.

Inhibitory CAR (i.e., iCAR) utilized intracellular domain of inhibitory receptors, PD-1 or CTLA-4, to disrupt T cell activation, proliferation, and cytokine release (Fedorov et al., 2013). It is designed as a dynamic safety guard to prevent ‘off-target’ toxicities when CAR-T cells are recognized to bind to normal cells. Compared with CTLA-4-based iCARs, PD-1-based iCARs showed more efficiency to inhibit numbers, proliferation and cytokine released of CAR-T cells.

Learning from iCAR, We designed a synthetic receptor with extracellular and transmembrane domains of IL-6R and intracellular domain of PD-1 (Fig.1 ④). The amino acid sequence of PD-1 was obtained with the Ensembl sequence, amino acids 191 to 289. With high level of IL-6, intracellular domain of PD-1 will be activated to inhibit downstream of T cell activation signals.

In humans, the concentration of IL6 is usually maintained at 0.00-2.90 pg/ml. With this in mind, we set up two sets of normal concentrations and three sets of abnormal concentrations to treat the cells that were then transferred. We found that the killing ability of Jurkat was enhanced after IL6 treatment, as shown in Figure 7. In the design concept of module1, we were expecting the expression of CAR to be inhibited at abnormal IL6 concentrations, thus reducing the side effects of killing; hence the problem with the system we designed. As the results of PD1 are shown in Figure 7, we can see that at high concentrations, PD1 successfully disrupts the signaling downstream of CAR-T, reducing the killing ability.

Although the experiment successfully proved that our system is problem-free, the activity of transfected Jurkat is not really high and the choice of dual promoter in the plot resulted in not high CAR expression, thus his killing is not very obvious. In addition, the concentration gradient in the normal group was set low and additional experimental groups should be set up in subsequent experiments.

In order to address the problem of high anti-aging CAR-T cost as reflected from human practices, we systematically analyzed the promising ways to solve the problem. The most widely used universal CAR-T (a type of CAR-T cell with TCR knockdown) is currently on the market. But given its potential host resistance problem and the technical challenges faced by secondary gene editing is beyond our capabilities (T cells start to be exhausted in about two weeks after isolation). We would like to explore a simpler approach, and another solution strategy is the in vivo production of CAR-T cells. Most of the previous studies have produced CAR-T cells in vivo by nanocarriers or liposomes, but they face problems of immunogenicity as well as organ delivery efficiency that are difficult to solve. Recently, exosomes have been widely studied as an excellent small molecule carrier, so we hope to solve this problem by exosomes. Also, considering that exosomes are secreted by cells, based on the idea of capsule cells in synthetic biology, we would like to develop a synthetic cell based on this and use it to produce CAR-T cells in vivo. Exosomes were first found in reticulocytes, while red blood cells, as one of the main components of blood transfusion, have a high degree of versatility among people of the same blood type. Therefore, we wanted to rely on erythrocytes as a medium (immature erythrocytes produce a large number of exosomes), and we considered erythrocytes as a potential engineered vector, considering their short self-life and the fact that they are not easily edited to become tumorigenic.