Targeted-Delivery Strategy of Engineered EV Functionalized By Anti-CD3/CD19 Bi-Specific T-Cell Engager in Treating B-Cell Malignancies

The incidence of B-cell malignancies in adults has increased in recent decades. However, the effects of current treatments for B-cell malignancies remain suboptimal due to the limited targeting ability and infiltration of therapeutic drugs. Several drug delivery systems were thus established and applied to overcome the limitations, including nanoparticles and extracellular vesicles (EVs). In contrast with conventional nanoparticles, EVs are characterized by high biocompatibility, easy to be engineered and modified, and low immunogenicity. EVs endow therapeutic development with unique and important characteristics. Here, we design a novel anti-CD3/CD19 bispecific T cell engager (BiTE) modified vesicle system. And we further validated its drug-loading capacity. We demonstrated that CD3/CD19 BiTE EV is a novel system that could mobilize T cells to target and kill CD19-positive cells and may synergistically activate immune cells in the tumor microenvironment (TME) by loading drugs.

To prepare BiTE EVs, we constructed a HEK293T cell line expressing anti-CD3/CD19 BiTE. We collected EVs at different time points after UV irradiation to optimize the process of producing EVs in HEK293T cells. Nanoparticle tracking analysis showed that the highest yield of EVs was achieved when the incubator was left for 4 hours after UV irradiation, sufficiently compensating for the previously published time-consuming protocol.

To evaluate whether BiTE EVs could activate T cells, we co-cultured primary T cells and BiTE EVs with different concentrations. Compared with anti-CD3/CD28 bead stimulation, 1nM or higher concentrations of BiTE EVs could effectively induce T cell expansion and activation without cytotoxicity. Next, we evaluate whether BiTE EVs could facilitate the anti-tumor effect of T cells. We co-cultured T cells and CD19-positive tumor cells in the presence of BiTE EVs at different concentrations. Accompanied by increased expression of CD107a and release of pro-inflammatory cytokines such as IFN - γ and TNF - α, BiTE EVs facilitated the anti-tumor effect of T cells in a CD19-positive cell-dependent and concentration-dependent manner.

Then, we verified the anti-tumor effect of BiTE EVs in vivo. The NCG mice were randomly divided into the WT EVs group, the BiTE EVs intermittent injection group with different administration frequencies and the BiTE EVs continuous injection group after being inoculated with luciferase-labeled Nalm6 cells on day 0. Starting from day 3, mice were intravenously injected with T cells every week along with BiTE EVs or WT EVs. Compared with the WT EVs group, the continuous injection group of BiTE EVs and the intermittent injection group of BiTE EVs showed significant in vivo anti-tumor effects without obvious side effects. The overall survival of mice treated with continuous and intermittent injection groups (twice a week) also significantly improved.

TME may hinder immune evasion and response to checkpoint blockade therapies. Pharmacological activation of the STING pathway is reported to create an immunologically hot TME. However, systemic delivery of STING agonists might lead to undesired off-target inflammatory responses. Here, STING agonists were selected to test the loading capacity of the BiTE EVs system. STING agonist was loaded into BiTE EVs (BiTE EVs@STING) by electroporation. The IRF Reporter (Luc) -THP-1 cell line was constructed to verify BiTE EVs@STING-mediated activation of the STING pathway. Compared with free STING agonists, BiTE EVs@STING activated the downstream pathway more effectively in the short-time treatment, possibly resulting from the significantly higher delivery efficiency. In vitro PBMC co-culture experiments also indicated that BiTE EVs@STING could effectively induce the activation of the dendritic cells and macrophages with up-expression of CD86. BiTE EVs@STING also promotes the killing ability of PBMCs on Nalm6.

In summary, anti-CD3/CD19 BiTE-modified EVs is a new delivery system capable of targeting and killing ability. The system has the potential to activate immune cells in the TME by loading drugs, thereby exerting a synergistic effect. The uniquely engineered EVs presented in this study possess great potential in treating B-cell malignancies and innovative strategies for future treatments of other malignancies.