133P - Cytotoxic efficacy of artificial vesicles obtained from CAR-T cells by ultrasonication

Background

The use of CAR-T therapy for treating solid tumors has encountered challenges due to the structure and immunological properties of the tumors. However, microvesicles, which represent cell-free biosimilar drugs, offer a promising solution to overcome these obstacles. One of the main issues faced in this approach is the small yield of microvesicles from the cell sources. This challenge can be addressed by incorporating various induction methods such as ultrasonication.

Methods

T lymphocytes and anti-CD19 CAR-T cells were activated by CD3/CD28 abs for 24 h, then ultrasonication was used to obtain artificial vesicles (AVs) – T-AVs and CAR-T-AVs, respectively. AVs were isolated by differential centrifugation and analyzed with scanning electron microscopy (SEM) using Merlin (Carl Zeiss). Then the cytotoxic efficacy of AVs against PC3M and PC3M(CD19+) tumor cells was tested using xCelligence real-time cell analysis (RTCA) system.

Results

SEM data: CAR-T-AVs (n=263) were predominantly smaller in size than T-AVs (n=376). The major population was represented by 51-75 nm particles with a narrow monodisperse peak. Whereas for T-AVs, most particles the size distribution was much wider, in the range of 75-200 nm. RTCA data: for PC3M control, neither added T/CAR-T cells nor their respective AVs had any significant effect on tumor cell proliferation. Opposite to that, for the PC3M(CD19+) tumor line, the cell index began to decrease 30 hrs after CAR-T-AVs addition – interestingly, with more pronounced effect in the wells with a lower concentration of vesicles (10 μg/ml). Both T-AVs and CAR-T-AVs showed a less pronounced cytotoxic effect at higher concentration (35 μg/ml), potentially due to nonspecific toxicity.

Conclusions

Ranging particles size distribution may indicate diverse dynamics in the cell membrane and intracellular content in response to activation of T/CAR-T cells and treatment with ultrasonic. Doze-dependence of CAR-T-AVs cytotoxicity and their detailed mechanism of action shall be addressed in the future studies. The work was funded by the Russian Science Foundation grant 23-24-00224 and performed in accordance with the Strategic Academic Leadership Program of the Kazan (Volga Region) Federal University (PRIORITET-2030).

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

The authors.

Funding

The work was funded by the Russian Science Foundation grant 23-24-00224 and performed in accordance with the Strategic Academic Leadership Program of the Kazan (Volga Region) Federal University (PRIORITET-2030).

Disclosure

All authors have declared no conflicts of interest.