Our experiments are composed of three main parts: anti-DPP4 CAR-T cells construction，IL-6 based negative feedback loop, and caffeine-based inducible switch. To prove the feasibility of our designs, we also build a cell line which highly expressed the DPP4, the target of our CAR-T cells. All the proof methods are widely used and reliable experiments.

From the NCBI, we got the DNA sequence of the human DPP4 sequence. The anti-DPP4 CAR scFv DNA sequence was got from the patent, CN101282994A. Other DNA sequences of the CAR including signal peptide, CD8 transmembrane domain, 4-1BB domain, and CD3-ζ domain was presented by our primary PI, He, Huang.

Then we assembled our CAR structure which was anti-DPP4 CAR scFv – CD8 transmembrane domain – 4-1BB domain – CD3-ζ domain. The leader sequence was added at the front of the CAR. The anti-DPP4 CAR scFv can recognize the DPP4 expressed on the senescent cells surface. Then it will activate the 4-1BB domain and CD3-ζ domain, which activates the CAR-T cells to proliferate and secrete the granzymes and perforins to kill the target cells.

We entrusted the production of our plasmids, DPP4 control plasmid and anti-DPP4 CAR plasmid to the GeneScript company. The plasmid PB-YEF63, which is presented by the He, Huang’s lab, was chosen as our plasmid backbone.

Both Jurkat and K562 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and maintained at 5% CO2 in air incubator at 37°C.

The DPP4 control plasmid was transfected into the K562 cells through the Advanced DNA RNA Transfection Reagent. The DPP4 plasmid was mixed with the transfection reagent as a ratio of 1:1 and added to the K562 cell medium.

The anti-DPP4 CAR plasmid was transfected into the Jurkat through electrotransfection and Lonza 4D-Nucleofector kit was used.

1. Jurkat cells were precultured to a density of 3 * 105 cells/ ml before Nucleofection.

2. The Supplement solution was premixed with Nucleofector Solution.

3. Jurkat cells were centrifuged at 90g for 10 min at room temperature and the supernatant was completely removed.

4. Then, the cells were resuspended in 4D-Nucleofector Solution.

5. Add the anti-DPP4 plasmid in the Jurkat cells suspension.

6. The mixtures were transferred into the Nucleocuvette Vessels.

7. The Vessels were then placed into the retainer of the 4D-Nucleofector X unit and the program CL120 was used.

8. The transfected Jurkat cells were incubated in the fresh medium at 37°C.

After transfection, the transfected K562 and Jurkat were collected and examined the positive rate through flow cytometry.

1. 100μL transfected K562 and Jurkat suspensions were collected.

2. Transfected K562 and Jurkat were centrifuged at 600 g for 3 min and then the supernatant was discarded.

3. The precipitation of cells was washed by 300 μL PBS for twice and resuspended by 100 μL PBS.

4. Then the transfected K562 and Jurkat were incubated with anti-DPP4 monoclonal antibodies which conjugated with APC and anti-human IgG monoclonal antibodies which conjugated with FITC, respectively.

5. The positive rate was detected by the flow cytometry.

The anti-DPP4 CAR transfected (CAR-T group) and non-transfected (control group) macrophages were co-cultured with transfected K562-DPP4 cells at a ratio of 1 : 5 in 96-wells plate. After 4h, 8h, 24h, and 48h of co-culture, we collected the cell suspension from CAR-T group and control group. The cells in both groups were incubated with anti-DPP4 monoclonal antibody which conjugated APC and the survival rate of the DPP4 positive K562 cells were detected by flow cytometry.

In addition, we repeated the experiment. We co-cultured 10^5 DPP4-K562 cells with and without 3*10^5 anti-DPP4 CAR-T cells, respectively, and added the appropriate amount of culture medium. And three sets of replicate experiments were set up. After that 200 µl of the co-culture system (with the addition of 0.2 test DPP4 antibody) was counted by flow collection at 24h.

Raji cell line

Raw 264.7 cell line

CAR-COSMO plasmid

Zetalife advanced DNA RNA transfection reagent

Totally four major plasmid fragments were constructed in the negative feedback loop, which were IL6R – Notch – Gal4KRAB (BBa K4175010), IL6-scFv – Notch – Gal4KRAB (BBa K4175008), IL6R – PD1 (BBa K4175011), and UAS-pSV40 – CAR19 (BBa K4175012).

IL6R sequences (amino acids 1 to 309 for BBa K4175010, and amino acids 1 to 386 for BBa K4175011) and PD1 (amino acids 191 to 289) were optimized from Ensembl, while IL6 scFv sequence was optimized from the patent, 201917040387 (Tan Hong Ji and Campana, 2021). The ‘notch’ part was the minimal core transmembrane domain of the wild-type notch, quoted from the published paper of Prof. Morsut (Morsut et al., 2016). Gal4KRAB and UAS-pSV40 part were optimized from parts posted by NMU_China 2021 (BBa_K511003 and BBa_K2446037). P2A part was optimized from BBa K1537016 designed by iGEM14_UESTC-GreenLife. Furthermore, our plasmid backbone, MND63, with CAR19 including CD8 transmembrane domain, CD8 hinge, 4-1BB domain, and CD3-ζ domain was presented by our primary PI, He, Huang. Each part was ligated according to the plasmid map below and entrusted to GenScript company for plasmid synthesis, using seamless cloning.