Epsilon-caprolactone-modified polyethylenimine as a genetic vehicle for stem cell-based bispecific antibody and exosome synergistic therapy

Abstract

Bispecific antibodies (BsAb) have gained significant momentum in clinical application. However, the rapid enzymolysis and metabolism of protein drugs usually induce short circulation in vivo, and developing an efficient protein delivery system still is a bottleneck. Mesenchymal stem cells (MSCs) have become an attractive therapeutic carrier for cancers. Genetic modification enables MSCs to express and secrete specific proteins, which is essential for therapeutic efficacy. However, efficient gene transfer into MSCs is still a challenge. In this study, we applied epsilon-caprolactone-modified polyethylenimine (PEI-CL) as an efficacy carrier for plasmid transfection into MSC that served as in situ 'cell factory' for anti-CD3/CD20 BsAb preparation. Herein, the PEI-CL encapsulates the minicircle plasmid and mediates cell transfection efficiently. Thus, the anti-CD3/CD20 BsAb is secreted from MSC and recruited T cell, resulting in highly sensitive cytotoxicity in the human B-cell lymphoma. Furthermore, these stem cells produce exosomes bearing MiR-15a/MiR-16, which could negatively regulate cancer's oncogenes BCL-2 for adjuvant therapy. Meanwhile, high immunologic factors like tumor necrosis factor-α and interferon-γ are generated and enhance immunotherapy efficacy. The engineered MSCs are demonstrated as an efficient route for BsAb production, and these bioactive components contribute to synergistic therapy, which would be an innovative treatment.

Keywords: bispecific antibody; exosome; polyethylenimine; stem cell.

Graphical abstract

Figure 1.

Characterization of PEI25K-CL/MCDNA genedelivery system. (A) The agarose gel electrophoresis analysis of free MCDNA and PEI25K-CL/MCDNA. (B) DLS results of PEI25K-CL/MCDNA. (C) Zeta potential of PEI25K-CL/MCDNA. (D) AFM height image and analysis of PEI25K-CL/MCDNA (N/P ratio is 20).

Figure 2.

Transfection of HucMSCs by PEI25K-CL. (A) Characteristic of HucMSCs by flow cytometry. (B) PEI25K-CL and lipofectamine 2000 mediated the GFP MCDNA transfection in HucMSCs. (C) The BsAbs in the culture medium of PEI25K-CL transfected HucMSCs measured by WB, and the secreted BsAb was evaluated by anti-Flag antibody. Lipo represents lipofectamine 2000-mediated transfection system. (D) The standard curve of His concentration by ELISA. (E) BsAb concentration was determined after 72 and 96 h of transfection. T-test. P = 0.0799. NS: no significance difference.

Figure 3.

(A) Osteogenic, adipogenic differentiations measured by alizarin red and oil red O staining. (B and C) myogenic differentiation ability of the transfected HucMSCs evaluated by WB experiment and confocal imaging. The scale bar is 100 μm. NC is short of negative control, which is the sample from the normal HucMSCs cells. (D) IVIS imaging of Raji–luciferase cells after incubating with CM and BsAb. (E) Statistic analysis of the cell activity ratio according to the luciferase imaging at different time points (E + T+BsAb vs E + T+CM, ***P < 0.0001 at 3 h and 6 h). (F) Specific cell lysis qualification of the Raji cells after LDH assay. E + T means the cell mixture solution of T cells (effector, E) and Raji cells (target, T). E + T+CM means cells added with HucMSC culture medium. E + T+BsAb represents BsAb incubated with E and T cells. (n = 3. Data show with mean ± SD. *P < 0.05, **P < 0.01).

Figure 4.

(A) Direct observation of the cytotoxicity by confocal imaging. Calcein-AM/PI staining. The live cells show green fluorescence, and the dead cells are red. (B) The interaction between CIK cells and Raji cells. PKH67 stained the CIK cells, while the anti-CD19 antibody labeled the Raji cells. (C) Statistic of the concentration of IFN-γ in the culture medium. (D) Statistic of the concentration of TNF-α in the culture medium. Data shown as mean ± SD, ***P < 0.001; **P < 0.01; *P < 0.05. NS: no significant difference.

Figure 5.

(A–C) Characterization of HucMSCs derived exosome: TEM and size distribution. (D and E) are the statistical data of the microRNA expression quantified by qPCR.

Figure 6.

Cytotoxicity of the exosome to the target cell Raji. (A) IVIS imaging of the luciferase–Raji cells after incubating with different concentrations of exosomes. (B) Statistic of the cell viability. ****P < 0.0001, compared with the 0 μg/100 μl treated group.

Figure 7.

(A) The scheme of the exosome-mediated cell apoptosis. (B) BCL-2 protein expression after treatment of the exosomes at 0 – 48 μg/100 μL. (C) Cytotoxicity effect of BsAb and exosome on Raji cells. Exo: exosomes. Data presented as percent of cell death. *P < 0.05, ****P < 0.0001.

Scheme 1.

Engineered mesenchymal stem cells secrete bispecific antibodies and exosomes for B-lymphoma therapy. (A) Cationic PEI25K-CL carrier delivers MCDNA into the MSCs to produce anti-CD3/CD20 bispecific antibodies and exosomes. (B) The synergistic therapeutic effect of the antitumor immune response and cell apoptosis. MSC-derived exosome loading microRNAs of miR-15a/miR-16 could deplete BCL-2 expression and then induce the cell apoptosis; on the other hand, the released BsAb recruit activated T cells and perform antibody-dependent cellular cytotoxicity.