We screened single-cell transcriptome sequencing data to identify novel aging-related targets for Chimeric antigen receptors (CAR) - T immunotherapy. Four heart-related datasets of mouse, rat and monkey were used for marker selection. Limma and DEGSeq2 were used for differential gene expression analysis. Genes significantly (p < 0.05) upregulated genes (LogFC > 0) in senescent samples were considered as potential markers. A Python-based web crawler was used to automate the filtering process of membrane protein based on UniProt database. A total of 63 genes (12, 51 upregulated in 4 and 3 datasets respectively) entered the follow-up screening. Expression of the potential markers should be relatively tissue-specific and low in T cells. Using UCSC, Human Protein Atlas and Mouse Cell Atlas of Zhejiang University databases, combined with results of literature analysis and other previous data set analysis, the final 6 potential targets were identified (Anxa3, Icam1, Vsir, Tspan8, Cyba, Fxyd5). Cell subset analysis was performed on the four datasets using Seurat 4.1.1 to obtain the expression level of each target in the cell subset. For synthetic biology, we expand the field of application of CAR-T immunotherapy while providing viable targets. We have also optimized the methodology for screening the targets applied to CAR-T therapies.

We developed two models, a model in which CAR-T kills target cells and an intracellular model in which CAR-T cells express CAR-T proteins. Based on our experiment results and data from previous studies about targeted CAR-T cell transfer that have been applied in clinical trials, like anti-CD19 CAR-T cell therapy, a computational model was built for predicting the therapeutic effect of the DDP4-targeted CAR-T cell. For the Intracellular CAR-T protein expression model, it was developed with parameters and models from previous studies to check whether the CAR-T gene we transfected was effective. All in all, the simulation of the modelling made up the in vivo experiment that has not been conducted, which serves as a good prediction for the effectiveness of the DPP4-targeted CAR-T cell and transcribed CAR-expressing genes, though the quantitative results provide limited reference since it’s a conceptual model. However, these models provide a basis for predicting the efficiency of the CAR-T killing system by adjusting different parameters.

We screened the Single-Chain Fragment Variable (scFv) for Dipeptidyl peptidase-4 (DPP4) and integrated it into the CAR structure. In addition, we have also innovated the insertion of antibody structures into CARs with engineering success. Our caffeine antibody (COSMO) has demonstrated low toxicity and high caffeine responsiveness to human cell lines in experiments. We have pioneered the use of negative feedback switches in CAR-T immunotherapy, which distinguishes the work of NMU-China in 2021. We designed the negative feedback switch to effectively modulate the killing activity of T cells to avoid side effects such as cytokine release syndrome.

We observed that the development of synthetic biology in immunology is much slower compared to, for example, microbes, probably due to the relative difficulty of engineering immune cells and the toxicity of exogenous proteins to eukaryotic cells. We innovatively applied Zetalife advanced DNA RNA Transfection Reagent and examined the transfection efficiency (>90%) in the Raw 264.7 cell line. The CAR-macrophages prepared in this way ensure the presence of stable high-expression CAR for 4-5 days, and if the transposon system is used, a stable high CAR-macrophage line can be formed. We believe that such an attempt is very helpful to advance synthetic immunology.

First, we innovatively expanded and publicized the application field of CAR-T immunotherapy, which has gained much attention in recent years for its high efficiency and innovative novel synthetic biology structures and has become a strategy for the efficient treatment of diseases in the clinic. In addition, we have boldly modified the structure of CAR by fusing COSMO into CAR and pioneered the study of applying two antibodies separately in the CAR structure. Finally, we designed the negative feedback switch, a protein that we designed from scratch ourselves, which was a bold attempt on our part while achieve engineering success. In addition, we discuss other strategies for engineering T cells in vivo in our future plans. This reflects our thinking and application of synthetic biology.