[논문]Antibody-secreting macrophages generated using CpG-free plasmid eliminate tumor cells through antibody-dependent cellular phagocytosis

Cha, Eun Bi; Shin, Keun Koo; Seo, Jinho; Oh, Doo-Byoung

doi:10.5483/bmbrep.2020.53.8.024

[국내논문] Antibody-secreting macrophages generated using CpG-free plasmid eliminate tumor cells through antibody-dependent cellular phagocytosis 원문보기

BMB reports, v.53 no.8, 2020년, pp.442 - 447

Cha, Eun Bi (Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) , Shin, Keun Koo (Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) , Seo, Jinho (Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) , Oh, Doo-Byoung (Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))

Abstract ▼ AI-Helper

The non-viral delivery of genes into macrophages, known as hard-to-transfect cells, is a challenge. In this study, the microporation of a CpG-free and small plasmid (pCGfd-GFP) showed high transfection efficiency, sustainable transgene expression, and good cell viability in the transfections of Raw 264.7 and primary bone marrow-derived macrophages. The non-viral method using the pCGfd vector encoding anti-EGFR single-chain Fv fused with Fc (scFv-Fc) generated the macrophages secreting anti-EGFR scFv-Fc. These macrophages effectively phagocytized tumor cells expressing EGFR through the antibody-dependent mechanism, as was proved by experiments using EGFR-knockout tumor cells. Finally, peri-tumoral injections of anti-EGFR scFv-Fc-secreting macrophages were shown to inhibit tumor growth in the xenograft mouse model.

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가설 설정

Fig. 4. Anti-EGFR scFv-Fc-secreting macrophages effectively inhibited tumor growth in vivo. (A) Schematic representation of the xenograft experiment.

제안 방법

The analysis of phagocytosis was performed using three tumor cell lines, A431, HT-29, and A673, representing high-, medium-, and low-EGFR-expressing cells, respectively (Supplementary Fig. 3). The anti-EGFR scFv-Fc-secreting RAW 264.
To confirm that phagocytosis occurred by binding anti-EGFR scFv-Fc to EGFR on the surface of the tumor cells, we constructed EGFR^KOA431 cells using the CRISPR-Cas9 system based on the pCGfd vector (pCGfd-gR-EGFR-Cas9-2A-GFP) for efficient transfection (Supplementary Fig. 4). As expected, antiEGFR scFv-Fc-secreting macrophages phagocytized very few EGFR^KOA431 cells, which was in contrast with the efficient phagocytosis of wild-type A431 cells (Fig.
In summary, in this study, we developed a CpG-free plasmid microporation method for the efficient transfection of macrophages and proved that these engineered macrophages were able to secrete antibodies and eliminate tumors via ADCP. As such, the findings presented here will contribute to the development of novel immune cell therapies for the treatment of cancer.
In this study, we employed a non-viral gene-delivery method using a CpG-free plasmid for the generation of macrophages that secrete anti-EGFR antibody.

데이터처리

Data represent the means ± standard deviation (SD), n = 3. Statistical significances were determined by two-way (C) or one-way (D) ANOVA, *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001.

성능/효과

Notably, the smallest plasmid, pCGfd-GFP, provided the highest efficiency. When the sustainability of GFP expression was analyzed for six days, the pCGfGFP and pCGfd-GFP microporations exhibited a longer GFP expression than did pcDNA3.1-GFP (Fig. 1C). The fact that the pCGfd-GFP plasmid displayed sustainability like that of pCGf-GFP indicated that the beneficial effect of plasmid size reduction was greater than the negative effect induced by the removal of two MARs.
Because unmethylated CpG sequences have been known to induce inflammatory responses and cell cytotoxicity (15), next, we determined whether a reduction in CpG sequences increases cell viability. When the viabilities were analyzed one day after the microporation (Fig. 1D), pCGf-GFP and pCGfd-GFP showed significantly higher viabilities (56% and 66%) than did pcDNA3.1- GFP (35%).

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DOI : 10.5483/BMBRep.2020.53.8.024
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