Targeting mesothelin receptors with drug-loaded bacterial nanocells suppresses human mesothelioma tumour growth in mouse xenograft models

Abstract

Human malignant mesothelioma is a chemoresistant tumour that develops from mesothelial cells, commonly associated with asbestos exposure. Malignant mesothelioma incidence rates in European countries are still rising and Australia has one of the highest burdens of malignant mesothelioma on a population basis in the world. Therapy using systemic delivery of free cytotoxic agents is associated with many undesirable side effects due to non-selectivity, and is thus dose-limited which limits its therapeutic potential. Therefore, increasing the selectivity of anti-cancer agents has the potential to dramatically enhance drug efficacy and reduce toxicity. EnGeneIC Dream Vectors (EDV) are antibody-targeted nanocells which can be loaded with cytotoxic drugs and delivered to specific cancer cells via bispecific antibodies (BsAbs) which target the EDV and a cancer cell-specific receptor, simultaneously. BsAbs were designed to target doxorubicin-loaded EDVs to cancer cells via cell surface mesothelin (MSLN). Flow cytometry was used to investigate cell binding and induction of apoptosis, and confocal microscopy to visualize internalization. Mouse xenograft models were used to assess anti-tumour effects in vivo, followed by immunohistochemistry for ex vivo evaluation of proliferation and necrosis. BsAb-targeted, doxorubicin-loaded EDVs were able to bind to and internalize within mesothelioma cells in vitro via MSLN receptors and induce apoptosis. In mice xenografts, the BsAb-targeted, doxorubicin-loaded EDVs suppressed the tumour growth and also decreased cell proliferation. Thus, the use of MSLN-specific antibodies to deliver encapsulated doxorubicin can provide a novel and alternative modality for treatment of mesothelioma.

Fig 1. Schematic representation showing the mechanism of ^{Anti-MSLN-BsAb}EDV_Dox nanocell mediated intracellular doxorubicin delivery.

Active binding of the EDV nanocells (yellow) to the MSLN cell-surface receptor (red) occurs via the BsAb. The EDV is lysed intracellularly to release the doxorubicin (purple) into the cell cytoplasm.

Fig 2. Anti-MSLN-LPS BsAb gene design, synthesis and binding to human mesothelioma cell lines.

(A) A BsAb comprising anti-MSLN scFv (red) and anti-LPS scFv (blue), connected by a glycine-serine linker (G4S). 6-HIS and C-MYC was included into the design for purification and detection purposes. Secretion peptide (SP) was included to enable the BsAbs to be secreted from CHO cells into medium. (B) SDS- PAGE gel showing, starting from left, SeeBlue pre-stained standard MW marker (Invitrogen) (Lane 1), Amatux-BsAb (Lane 2) and HN1-BsAb (Lane 3) purified products (Both MWs are 56 kDa). Flow cytometry was used to test the anti-MSLN designed BsAbs and an anti-EGFR control BsAb (ABX-EGF-BsAb) binding to human mesothelioma cell lines, (C) H226 and (D) MSTO-211H. Binding of anti-CD3-BsAb (black line), ABX-EGF-BsAb (red line), Amatux-BsAb (green line) and HN1-BsAb (orange line) to cells was detected using FITC-conjugated anti-c-myc antibody.

Fig 3. BsAb targeting and internalisation of EDV_Dox to MSLN on H226 cells.

Internalisation was detected using confocal imaging of H226 cells incubated with Amatux-BsAb targeted AF-488-EDV nanocells (Green: Ex 488; Em 490–540) loaded with doxorubicin (Purple: Ex 488; Em 540–590) at 3 hr (A) and 24 hr (B) timepoints (Scale bars 5 and 10 μm, respectively). H226 cell membranes were pre-stained with anti-EGFR-AF-647 (Red: Ex 633; Em 640–750). Specificity of ^Non-targetedEDV_Dox (red line), ^mEpCAMEDV_Dox (black line) and ^AmatuxEDV_Dox (green line) binding to human mesothelioma cell lines, H226 (C) and MSTO-211H (D), and dose-response assay of ^AmatuxEDV_Dox on H226 (E), MSTO-211H (F) and CT26 (G) were excuted by flow cytometry using anti-LPS AF-488 and 488 nm laser and 530/30 nm filter.

Fig 4. internalisation of ^AmatuxEDV_Dox apoptosis induction.

The induction of apoptosis due to release of doxorubicin internally was measured using Annexin V-FITC staining, on untreated cells (1), cells treated with ^AmatuxEDV (2), ^Non-targetedEDV_Dox (3), or ^AmatuxEDV_Dox (4). Fluorescence was measured using 488 nm laser and 530/30 nm filter. The red dot plot represents Annexin V-FITC positive cells and the green dot plot represents Annexin V-FITC negative cells.

Fig 5. Effect of ^AmatuxEDV_Dox on xenograft models.

(A) Xenograft implanted mice were treated with either saline as a control, or EDV variants ^AmatuxEDV, ^Non-targetedEDV_Dox or ^AmatuxEDV_Dox all at a dose equal to 1 x 10⁹ EDVs at different time points, indicated with a red triangle. The average tumour size at the start is ~110 mm³. (B) The mice were weighed twice a week throughout the treatment. Mean ± SEM is shown. (*** = p < 0.001).

Fig 6. Immunohistochemical staining of paraffin embedded mesothelioma xenograft tumour samples.

(A) Malignant mesothelial cells showed strong and diffuse staining by anti-human pKi67 antibody. Tissue stained with secondary antibody only showed no staining. Scale bar 200 μm. (B) The percentage of Ki67-positive cells were calculated blindly from tumour tissue sections obtained from three different mice representing each group (Mean ± SD). (C) Tumour necrotic areas in the tissue sections are shown by reduced H&E staining. Representative tissue sections for the four treatment groups show necrotic areas (N) (absence of nuclear staining). Scale bar 500 μm. (D) The percentage of necrosis was calculated blindly using ImageJ from tumour tissue sections obtained from three different mice representing each group (Mean ± SD). (* = p < 0.05, ** = p < .01 and *** = p < 0.0005).