scFv(GPC3)-CD28-41BB-CD3zeta CAR-T Lentivirus

One of the key technological advantages of lentiviral platforms is their ability to stably integrate chimeric antigen receptor (CAR) transgenes into the host cell genome, typically primary human T cells or Jurkat cell lines. This integration ensures the long-term genetic expression of the anti-GPC3 CAR during cell proliferation, which is crucial for maintaining antitumor activity in adoptive cell therapy. Furthermore, these lentiviral particles are pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G), resulting in broad tropism and the ability to transduce various mammalian cell types at high titers with low toxicity. From a safety perspective, third-generation CAR-T systems employ a segregated genome design, placing essential viral packaging genes on separate plasmids and modifying long terminal repeats (LTRs) with self-inactivating sequences (SINs). This configuration significantly reduces the risk of generating replicative lentiviruses (RCLs) and minimizes the possibility of insertional mutations. Moreover, the inclusion of three intracellular signaling domains—CD28, 4-1BB, and CD3ζ—in the lentiviral vector enables the preparation of third-generation CAR-T cells. Compared to second-generation CAR-T cells, these cells exhibit greater persistence, superior metabolic properties, and stronger cytotoxicity because they utilize the rapid activation kinetics of the CD28 domain and the long-term survival and cell exhaustion-delaying signals provided by the 4-1BB domain.

The primary application of the scFv(GPC3)-CD28-41BB-CD3zeta CAR-T lentivirus is in the development of immunotherapies targeting Glypican-3 (GPC3)-positive malignancies, particularly hepatocellular carcinoma (HCC). Since GPC3 is highly expressed in HCC but not in healthy adult liver tissue, this lentivirus serves as a precise tool for constructing CAR-T cells, enabling them to specifically recognize and eliminate malignant cells. In preclinical studies, these lentiviral particles were used to transduce human peripheral blood mononuclear cells (PBMCs) to evaluate the therapeutic efficacy of third-generation CAR constructs. Researchers used these transduced cells to conduct various in vitro experiments, such as cytotoxicity assays based on luciferase or LDH release, to assess the cytotoxic efficacy of CAR-T cells against GPC3-expressing target cell lines (such as HepG2 or Huh7). Furthermore, these particles are crucial for establishing in vivo xenograft mouse models, in which the persistence and infiltration of third-generation CAR-T cells in the complex solid tumor microenvironment can be investigated in depth. Because the virus contains both CD28 and 4-1BB co-stimulatory domains, it is an ideal candidate vector for comparative studies aimed at overcoming the immunosuppressive properties of the solid tumor microenvironment.