Chimeric antigen receptor (CAR) therapeutic cells have shown remarkable promise in treating leukemia and lymphoma. However, generating CAR T or NK cells in vivo for solid tumors faces challenges: current approaches engineer cells in blood, spleen, or bone marrow, but these cells must overcome extensive physiological barriers to infiltrate solid tumors, often losing their cytotoxic potency along the way.
A research team led by Prof. Yaping Li and Prof. Dangge Wang has developed an elegant solution to this problem. They designed a GRM4-targeted, tumor-penetrable lipoplex that can generate NKG2D CARs directly within triple-negative breast cancer, eliminating the need for cells to travel long distances to reach their target.
The researchers modified lipoplexes with a tumor-penetrating peptide (octa-arginine, R8) and O-Phospho-L-serine to target GRM4, a receptor highly expressed in breast cancer tissues and immune cells within the tumor microenvironment. The lipoplexes carry plasmid DNA encoding NKG2D CAR and interleukin-15. To ensure tumor-specific CAR generation, the team incorporated a clever safety mechanism: a promoter containing nine consecutive hypoxia-responsive elements (9×HREs) and an oxygen-dependent degradation domain. This design ensures that CARs are only expressed under the hypoxic conditions typically found in solid tumors, not in healthy tissues. The IL-15 sequence is not designed with a hypoxia-sensitive promoter, allowing sustained stimulation of immune cells.
The GRM4-targeted lipoplexes (R8L-NI@Lipo) showed remarkable tumor accumulation and penetration capabilities. In mouse models, the lipoplexes diffused from blood vessels into the core regions of tumors, unlike non-targeted versions that remained trapped at the periphery. Importantly, the hypoxia-inducible design tightly controlled CAR expression. At oxygen levels of 5% or higher, NKG2D expression was undetectable; but under hypoxic conditions (1% O 2 or lower), expression increased by 2.48-fold. This dual-control strategy significantly reduced off-target CAR expression in healthy organs such as the heart, liver, spleen, lung, and kidney.
When tested in mouse models of triple-negative breast cancer, the R8L-NI@Lipo treatment increased infiltration of CD8⁺ T cells by 3.33-fold, enhanced NK cell activation and cytotoxic function, repolarized tumor-associated macrophages from M2 to M1 phenotype, inhibited tumor growth and extended median survival from 22.5 days to 42.5 days.
This study provides an attractive precision strategy to engineer CARs in solid tumors with enhanced safety. By integrating active targeting, tumor penetration, and hypoxia-inducible expression, the approach bypasses the trafficking hurdles that typically limit CAR therapy efficacy in solid tumors. The platform could potentially be adapted for other human cancers in future studies.
Science Bulletin
Experimental study