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. 2016 Mar;11(3):295-303.
doi: 10.1038/nnano.2015.292. Epub 2015 Dec 21.

In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer

P H Lizotte  1 , A M Wen  2 , M R Sheen  1 , J Fields  1 , P Rojanasopondist  1 , N F Steinmetz  2   3   4   5   6 , S Fiering  1   7   8
Affiliations

In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer

P H Lizotte et al. Nat Nanotechnol. 2016 Mar.

Abstract

Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The 'in situ vaccination' immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic antitumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from cowpea mosaic virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic antitumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the antitumour immune response. CPMV also exhibited clear treatment efficacy and systemic antitumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer.

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Conflict of interest statement

Competing interests

P.H.L., A.M.W., N.F.S., and S.F. have applied for patent protection for the immunotherapeutic use of eCPMV.

Figures

Figure 1
Figure 1. eCPMV nanoparticles are inherently immuonogenic
a, Bone marrow-derived dendritic cells (BMDCs) exposed to eCPMV produce elevated levels of pro-inflammatory cytokines in vitro. b, Thioglycollate-elicited primary macrophages also secrete significantly elevated levels of the same panel of cytokines. Both cell types (n = 6/group) were cultured for 24hr with 20μg eCPMV (dark gray bars) and cytokine levels were analysed using a multiplexed luminex array. Data for bar graphs calculated using unpaired Student’s t-test with p <0.05 as *, p <0.01 as **, and p <0.001 as ***.
Figure 2
Figure 2. eCPMV inhalation induces dramatic changes in lung immune cell composition and cytokine/chemokine milieu in mice bearing B16F10 lung tumours
a, Representative FACS plots pre-gated on live CD45+ cells of non-tumour-bearing mice treated with PBS (top left) or eCPMV (top right) and B16F10 lung tumour-bearing mice treated with PBS (bottom left) or eCPMV (bottom right). B16F10 mice were treated on day 7 post-B16F10 IV injection. Lungs were harvested 24hr after intratracheal injection of PBS or 100ug eCPMV. Labeling indicates (i) quiescent neutrophils, (ii) alveolar macrophages, (iii) monocytic MDSCs, (iv) granulocytic MDSCs, (v) tumour-infiltrating neutrophils, and (vi) activated neutrophils. Numbers beside circled groups are % of CD45+ cells. Arrows indicate TINs (blue) and CD11b+ activated neutrophils (red). Gating strategies available in Supplemental Fig. 3. b, Changes in innate cell subsets induced by eCPMV inhalation in tumour-bearing mice (n = 5/group) are quantified as a percentage of CD45+ cells (top) and total number of cells (bottom) as presented in panel a. c, Representative histograms for TINs (blue), activated neutrophils (red), alveolar macrophages (green), and monocytic MDSCs (orange) indicating mean fluorescence intensity (MFI) uptake of Alexa488-labeled CPMV, class-II, and CD86 activation markers. d, Lungs of B16F10 lung tumour-bearing mice (n = 5/group) exhibited elevated levels of pro-inflammatory cytokines and chemoattractants when treated with eCPMV as in panel a. Data for bar graphs calculated using unpaired Student’s t-test with p <0.05 as *, p <0.01 as **, and p <0.001 as ***.
Figure 3
Figure 3. eCPMV inhalation reduces formation of B16F10 metastatic-like lung tumours
a, Schematic of experimental design. b, Photographic images of lungs from eCPMV- and PBS-treated B16F10 tumour-bearing mice on day 21 post-tumour challenge. c–d, B16F10 lung metastatic-like tumour foci were quantified both by number in c or by qRT-PCR assay for melanocyte-specific Tyrp1 mRNA expression in d (n = 8 eCPMV, 7 PBS). Data for bar graphs calculated using unpaired Student’s t-test with p <0.05 as *, p <0.01 as **, and p <0.001 as ***.
Figure 4
Figure 4. eCPMV treatment efficacy in B16F10 lung model is immune-mediated
a, eCPMV inhalation did not significantly affect tumour progression when mice lack Il-12 (n = 7 eCPMV, 8 PBS). b, Treatment efficacy was also abrogated in the absence of Ifn-γ (n = 5/group). c, NOD/scid/Il2R-γ−/− mice lacking T, B, and NK cells also failed to respond to eCPMV inhalation therapy (n = 5/group). d, Depletion of neutrophils with Ly6G mAb abrogates treatment efficacy (n = 5/group). Data for bar graphs calculated using unpaired Student’s t-test with p <0.05 as *, p <0.01 as **, and p <0.001 as ***.
Figure 5
Figure 5. eCPMV immunotherapy is successful in metastatic breast, colon, and ovarian carcinoma models
a, Mice challenged with 4T1 breast tumours and intratracheally injected with PBS rapidly developed (IVIS images) and succumbed (Kaplan-Meier) to metastatic lung tumours beginning on day 24 post-surgical removal of primary tumour, whereas tumour development was delayed and survival significantly extended in mice receiving intratracheal injection of eCPMV (n = 8 eCPMV, 5 PBS). b, Mice bearing intradermal flank CT26 colon tumours also responded to direct injection of eCPMV (arrows indicate treatment days) with significantly delayed growth when compared to PBS-injected controls (n = 5/group). c, eCPMV also proved successful as a therapy for ID8-Defb29/Vegf-A ovarian cancer-challenged mice, significantly improving survival when injected IP relative to PBS-injected controls (n = 4 eCPMV, 11 PBS). eCPMV-treated mice displayed no visible ascites on day 42 post-challenge while PBS-treated controls had reached endstage criteria. Survival experiments utilised the log-rank Mantel-Cox test for survival analysis and flank tumour growth curves were analysed using two-way ANOVA, with p <0.05 as *, p <0.01 as **, and p <0.001 as ***.
Figure 6
Figure 6. eCPMV induces systemic, durable anti-tumour immunity
a–b, Mice bearing intradermal flank B16F10 tumours directly injected with eCPMV (arrows indicate treatment days) showed noticeably delayed tumour progression relative to PBS-injected controls (n = 8 eCPMV, 6 PBS). c, Half of eCPMV-treated mice experienced complete elimination of primary tumours (n = 8 eCPMV, 6 PBS). d, The majority of mice cured of primary tumours by eCPMV treatment and re-challenged on the opposite flank 4 weeks later failed to develop new tumours (n = 4/group). Survival experiments utilised the log-rank Mantel-Cox test for survival analysis and flank tumour growth curves were analysed using two-way ANOVA, with p <0.05 as *, p <0.01 as **, and p <0.001 as ***.
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