For in vivo CAR T cell generation, these viral vectors and nanocarriers can be further engineered to specifically target T cells by incorporating T cell-targeting ligands. These next-generation delivery systems ensure that genetic payloads are delivered with high precision, thereby maximizing the therapeutic index and minimizing risks associated with systemic, off-target biodistribution.
Selecting the optimal cell-surface receptor for in vivo targeting is a critical design choice that determines the specificity and potency of the resulting CAR-T product. In vivo T-cell engineering via targeted delivery platforms leverages highly expressed lineage markers, such as CD3, CD8, CD28, CD2, CD5, and CD7 [1], and delivery strategies utilizing some of these receptors have already made their way to the clinic. Recently, Umoja developed an anti-CD3 scFv-modified lentivirus (LV) for generating anti-CD19 CAR in CD3+ T cells in vivo, exhibiting selective expansion and achieving elimination of B-cell malignancies in xenograft mouse models [2].
Research has also shown significant progress in delivering CAR constructs using targeted nanocarriers. In recent work, CD3-targeted lipid nanocarriers (tLNPs) loaded with plasmids containing interleukin 6 short hairpin RNA and CD19-CAR genes selectively targeted circulating T cells. This approach increased CAR expression and extended anti-tumor effects of the resulting CAR T cells in leukemic mice for up to 41 days post-administration, comparable to ex vivo T cell therapy [3]. In a separate study, researchers utilized CD3-targeted nanoparticles to deliver leukemia-specific CAR genes to T cells in a leukemic mouse model. Four hours after tail vein injection, these T cell-targeted nanoparticles were found to have successfully delivered CAR genes to circulating T cells, which resulted in tumor regression and enhanced survival of up to 5 days in a 120-day follow-up period [4].
Similarly, Hunter et al. used CD8-tLNPs to reprogram CD8+ T cells with CD19 CAR constructs in both healthy donor and autoimmune patient samples [5]. In vivo dosing resulted in tumor control in humanized mice and B-cell depletion in cynomolgus monkeys, with the cyno reconstituted B cells exhibiting a predominantly naïve phenotype, indicative of an immune system reset [5].
In another example, Parayath et al. leveraged CD3-targeted nanoparticles to deliver a CD19 CAR (1928z). In vivo reprogramming of T cells in immunodeficient mice bearing human leukemia tumors led to leukemia regression and improved survival, comparable to adoptive T‑cell therapy.
