Liver cancer is the sixth most common cancer worldwide and ranks among the top three in terms of mortality. Chimeric Antigen Receptor (CAR)-T cells, which have shown good therapeutic effects in hematologic tumors, struggle to penetrate into the interior of liver cancer solid tumors. On the other hand, CAR-macrophages (CAR-M) capable of infiltrating liver cancer solid tumors are limited by tumor antigen heterogeneity and the immunosuppressive tumor microenvironment, resulting in suboptimal therapeutic efficacy. This year, our AFMU-China team has designed synthetic Notch system (SynNotch)-based engineered macrophages for liver cancer therapy, which we call SYNERGY (also be referred to as Syn-M when describing experimental results). SYNERGY, through our carefully designed signal input and output components, has successfully overcome the two major challenges previously mentioned for CAR-macrophages, demonstrating strong efficacy and safety. It holds promise to provide new insights and potential approaches for liver cancer treatment. Furthermore, we are also exploring the use of LNP technology to reduce the treatment cost of SYNERGY, thereby benefiting more patients and potentially transforming the landscape of liver cancer care.

Liver cancer is the sixth most common tumor in the world, and the mortality rate is among the top three. The incidence and mortality of liver cancer are still increasing worldwide, and it is expected that the annual new cases of liver cancer will increase by 55% between 2020 and 2040. By 2040, 1.4 million people will be diagnosed with liver cancer, and 1.3 million people will die from liver cancer worldwide. According to data statistics, there were 431, 383 new cases of liver cancer and 412, 216 deaths in China in 2022. It has brought heavy medical and economic burden. These figures highlight the great social significance of studying the cellular and molecular mechanisms of liver cancer pathogenesis and establishing new strategies for intervention and reversal to reduce the incidence and mortality of liver cancer.

The treatment of liver cancer aims to reduce mortality and improve the quality of life of patients. The treatment methods include surgical treatment, locoregional treatment and systemic treatment. Surgical treatment includes liver resection and liver transplantation. The former is the preferred treatment for HCC in patients without cirrhosis, and the latter is theoretically the best treatment option. Locoregional treatment includes thermal ablation, interventional therapy, and radiotherapy. Systemic treatment includes targeted therapy, immunotherapy and other strategies. Sorafenib and regorafenib significantly improve the survival rate of patients with advanced liver cancer and those who transitioned to advanced liver cancer after the failure of other treatments. Although great progress has been made in the treatment of liver cancer, it still shows obvious shortcomings, such as few opportunities for surgery, high recurrence rate, lack of liver transplant donors, limited systemic treatment options, low response rate of immunotherapy and low five-year survival rate (only 18%), etc. Therefore, it is necessary to develop a more effective treatment strategy for liver cancer.

In recent years, new therapies for liver cancer based on gene editing and tumor microenvironment regulation are gradually entering the clinical vision, providing new hope for breaking through the current treatment bottleneck. However, new cancer therapies, such as targeted drugs and immune checkpoint inhibitors, still rely on the patient's exhausted immune system. To truly "rewrite" the course of advanced cancer, we must first "rebuild" immune cells that can continuously recognize, kill, and remember tumor antigens. Therefore, adoptive cell therapy represented by CAR-T, CAR-M, and CAR-NK has been rapidly applied to clinical treatment.

In 2017, CAR-T cells were officially put into clinical treatment, among which adoptive T cell therapy was the first to achieve grade Ⅰ/Ⅱ clinical evidence in the treatment of hepatocellular carcinoma, but adoptive T cell therapy still faces multiple challenges such as difficult invasion in the treatment of hepatocellular carcinoma. Therefore, CAR-M emerged. Even though it has the advantages of invasion and phagocytosis, CAR-M therapy still faces the following defects in liver cancer treatment.

• Nearly all types of liver cancer consist of heterogeneous subpopulations of cancer cells exhibiting diverse phenotypes and genotypes. In other words, within the same liver tumor, different cancer cells express varying levels of distinct tumor-associated antigens. This tumor antigen heterogeneity severely impairs the ability of CAR-M to detect liver cancer cells and significantly diminishes therapeutic efficacy.
• At the onset of therapy, CAR-M exhibit an anti-tumor M1 phenotype. However, they are susceptible to "reprogramming" or "acclimatization" by the tumor microenvironment - factors such as IL-10, TGF-β, and hypoxia can drive their anti-polarization from the anti-tumor M1 state toward the pro-tumor M2 phenotype. This immunosuppressive tumor microenvironment supports the maintenance of the liver cancer 's malignant biological behaviors.

In order to overcome the above-mentioned problems of tumor antigen heterogeneity and immunosuppressive microenvironment in traditional CAR-M therapy, our team (AFMU-China 2025) has conducted in-depth research on the pathogenesis and immune escape characteristics of liver cancer, carried out multi-dimensional communication with clinical experts, immunologists and other multidisciplinary experts, and focused on synthetic biology technology. Ultimately, we decided to introduce a carefully designed synthetic Notch system into the macrophage chassis, resulting in our solution: SYNERGY. SYNERGY can be activated upon receiving a specific INPUT signal, thereby triggering a defined OUTPUT response.

• The key component of SYNERGY's INPUT module is an anti-SLC17A2 scFv independently developed by our team. Upon binding to SLC17A2—a protein that is highly and specifically expressed on the surface of hepatocytes—SYNERGY is activated. This design ensures that SYNERGY can be triggered within the liver microenvironment without losing therapeutic efficacy due to its inability to recognize heterogeneous liver cancer antigens.
• The key component of SYNERGY's OUTPUT module is the P65–SIRPα shRNA bicistronic system, independently developed by our team. Upon activation, SYNERGY simultaneously overexpresses P65 and SIRPα shRNA. P65 activates the NF-κB pathway, reprogramming SYNERGY into an anti-tumor M1-like macrophage, while SIRPα shRNA disrupts the "don't eat me" signal used by liver cancer cells to evade immune detection. These two mechanisms work synergistically to overcome the immunosuppressive tumor microenvironment.

After preliminary validation of SYNERGY’ s efficacy, we realized that its high treatment cost might be prohibitive for many patients. Leveraging our team’ s interdisciplinary expertise, spanning biomedical engineering, pharmacy, clinical medicine, immunology, and nanomaterials, we comprehensively reviewed the current landscape and key bottlenecks in liver cancer therapy and systematically evaluated the integrative potential of three core technologies: lipid nanoparticles (LNPs), gene editing, and macrophage engineering. Ultimately, we decided to adopt LNP as the primary delivery platform to enable in situ gene editing of SYNERGY.

Compared with traditional ex vivo cell engineering, LNPs offer significant advantages, including rapid delivery, transient expression, no requirement for viral vectors, and ease of large-scale manufacturing, providing a safer and more cost-effective solution for cell therapy. In this project, we designed LNPs targeting tumor-associated macrophages (TAMs) in hepatocellular carcinoma. Cell-specific recognition was achieved by conjugating an M2 macrophage-targeting peptide onto the LNP surface, and the LNPs were loaded with the full set of plasmids encoding the SYNERGY system. This enables in situ reprogramming of pro-tumorigenic TAMs into anti-tumor SYNERGY macrophages. This design overcomes the complex workflow of ex vivo cell expansion and reinfusion, laying a technical foundation for a ‘single-shot’ immunotherapy for liver cancer.

Leveraging the modularity and programmability of the LNP platform, the Synergy system holds promise for broader applications in a range of liver diseases. While the current project focuses on hepatocellular carcinoma, its core mechanism, liver-specific activation and remodeling of the immune microenvironment, is equally applicable to chronic liver conditions such as hepatitis, liver fibrosis, and cirrhosis. By swapping the downstream transcriptional elements of the SynNotch system, for example, replacing anti-tumor genes with anti-fibrotic factors such as IFN-γ or TGF-β inhibitors, precise intervention tailored to distinct pathological states of the liver can be achieved.

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