Anti-HER2 scFv-Directed Extracellular Vesicle-Mediated mRNA-Based Gene Delivery Inhibits Growth of HER2-Positive Human Breast Tumor Xenografts by Prodrug Activation

Abstract

This paper deals with specific targeting of the prodrug/enzyme regimen, CNOB/HChrR6, to treat a serious disease, namely HER2⁺ human breast cancer with minimal off-target toxicity. HChrR6 is an improved bacterial enzyme that converts CNOB into the cytotoxic drug MCHB. Extracellular vesicles (EV) were used for mRNA-based HchrR6 gene delivery: EVs may cause minimal immune rejection, and mRNA may be superior to DNA for gene delivery. To confine HChrR6 generation and CNOB activation to the cancer, the EVHB chimeric protein was constructed. It contains high-affinity anti-HER2 scFv antibody (ML39) and is capable of latching on to EV surface. Cells transfected with EVHB-encoding plasmid generated EVs displaying this protein ("directed EVs"). Transfection of a separate batch of cells with the new plasmid, XPort/HChrR6, generated EVs containing HChrR6 mRNA; incubation with pure EVHB enabled these to target the HER2 receptor, generating "EXO-DEPT" EVs. EXO-DEPT treatment specifically enabled HER2-overexpressing BT474 cells to convert CNOB into MCHB in actinomycin D-independent manner, showing successful and specific delivery of HChrR6 mRNA. EXO-DEPTs-but not undirected EVs-plus CNOB caused near-complete growth arrest of orthotopic BT474 xenografts in vivo, demonstrating for the first time EV-mediated delivery of functional exogenous mRNA to tumors. EXO-DEPTs may be generated from patients' own dendritic cells to evade immune rejection, and without plasmids and their potentially harmful genetic material, raising the prospect of clinical use of this regimen. This approach can be used to treat any disease overexpressing a specific marker. Mol Cancer Ther; 17(5); 1133-42. ©2018 AACR.

Figure 1

Residual survival of cells after CNOB (15mM) and ChrR6 (50mg/mL) treatment (for 24 hours) in vitro. Cell viability was determined by MTT assay. Data are presented as percent survival (hashed bar) compared to untreated controls (solid dark bar) of the corresponding cells. MCF7 cells express low and BT474 cells high levels of the HER2 ligand (see text). HER2-overexpressing counterpart of MCF7 cells (“MCF7/ErbB2”) were included, as were Trastuzumab-resistant BT474 cells (“BT474/HER2-Res”) ***p<0.001 as compared to untreated control of the corresponding cell line.

Figure 2

HER2 receptor targeting ML39 chimeric protein (EVHB), and EV NanoSight analysis A. Schematic of EVHB: LS (blue), Lactadherin leader sequence for export of EVHB across the membrane; ML39 scFv (green), high affinity (Kd=10⁻⁹M) HER2-targeting moiety; lactadherin C1C2 domains (red), which enable EVHB to bind to the EV surface; His (orange), His-tag for purification; the predicted molecular weight of EVHB is 68kDA (Supplementary Fig. S1). B. EV NanoSight analysis. C. Western blots. Protein extracts of whole cells of HEK293 cells transfected with pEVC1C2HER plasmid or EVs generated by them; cells transfected with the empty plasmid (p6mLSC1C2) were used as control; the 68kDa band is seen only in the transfected cells and EVs generated from them. D. Predicted protein structure of EVHB. Color scheme: yellow, scFv antibody; red, leader sequence; blue, C1C2 domains.

Figure 3

Specificity of directed-EV (displaying EVHB) binding to HER2 receptor. A. ELISA detection of this activity: Orange bar, EVs obtained from pEVC1C2HER plasmid-transfected HEK293 cells; dark brown bar, EVs obtained from non-transfected HEK293 cells incubated with pure EVHB– these show greater binding capability (see text for details). No signal resulted when naïve-EVs (isolated from non-transfected HEK293 cells not incubated with EVHB), or PBS. Bars represent average value ± SD (n=3). *** p<0.001 as determined by t-test between groups as indicated. B. Schematic representation of EVHB display by EVs: “Transfection” shows EVs from HEK 293 cells transfected with pEVC1C2HER plasmid; “Reconstitution” indicates EVs obtained from non-transfected cells after incubation with pure EVHB. Yellow circles represent EVs, and red squiggles with green heads represent EVHB; C. Visualization of directed-EV binding specifically to HER2+ve cells. Representative fluorescent and phase contrast images of corresponding regions are shown: CFSE-labeled (green) directed-EVs bind to BT474 cells and not to MCF7 cells. D. Directed-EV binding to cells as determined by flow cytometry. Left panel: fluorescence shift caused by the indicated cell types (or mixture). The shift due to SKBR3 cells is arbitrarily assigned a value of 1 (see Results). Right panel: Quantification of the relative shifts based on the data of the left panel – binding is specific to HER2+ve cells.

Figure 4

Loading of EVs with HChrR6 mRNA and EXO-DEPT functionality in vitro and in vivo. A. Design of XPort/HChrR6 plasmid showing key features involved in mRNA packaging into EVs; see text for details. B. qPCR results showing successful loading of EVs with HChrR6 mRNA. Endogenous EV miR-16 level was determined as control. (The Ct value of mRNA corresponds to 2×10⁻⁴ copy/EV.) C. In vitro effectiveness of EXO-DEPT EVs. BT474 cells (3×10⁴) treated with 8×10⁸ EXO-DEPT EVs generated MCHB fluorescence upon CNOB treatment (dark brown bar); naive EV alone (dark blue bar) or loaded but non-directed EVs (not displaying EVHB) (orange bar) show only background fluorescence upon CNOB treatment. D. MCHB fluorescence normalized to cell viability. BT474 cells treated with EXO-DEPT EVs and CNOB generate MCHB fluorescence (hashed brown bar), which is not affected by the presence of actinomycin D (hashed blue bar) but is eliminated in the presence of cyclohexamide (CHX). See text for further details. Bars represent average value ± SD (n=3). *** p<0.001, ** p<0.01 as compared between groups as indicated. E. Administration schedule of EVs and CNOB for in vivo test of the effectiveness of EXO-DEPT EVs. Nu/nu mice implanted with orthotopic BT474 tumors were used (the number of EVs administered delivered 4×10⁵ copies of the HChrR6 mRNA per injection; CNOB per injection was 3.3 mg/Kg). F. Plot of average tumor volume recorded twice a week for the indicated treatment groups. G. Rate of tumor growth calculated from slopes of linear regression shown in Box and Whisker plot for each treatment group. Statistical analysis of linear regression slopes between groups was performed by two-samples, two-sided t-test, and confirmed by Tukey’s honest significance difference test as post-hoc. *** p<0.001, ** p<0.01, * p<0.05 as compared between groups as indicated. Further statistical analysis is provided in Supplementary Materials and Methods.