In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer

Journal name:
Nature Nanotechnology
Volume:
11,
Pages:
295–303
Year published:
DOI:
doi:10.1038/nnano.2015.292
Received
Accepted
Published online

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.

At a glance

Figures

  1. eCPMV nanoparticles are inherently immuonogenic.
    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 24 h with 20 µg 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. *p < 0.05; **p < 0.01; ***p < 0.001.

  2. eCPMV inhalation induces dramatic changes in lung immune cell composition and cytokine/chemokine milieu in mice bearing B16F10 lung tumours.
    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 24 h after intratracheal injection of PBS or 100 ug 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 Supplementary 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, activated neutrophils, alveolar macrophages and monocytic MDSCs, indicating mean fluorescence intensity (MFI) uptake of Alexa488-labelled 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. *p < 0.05; **p < 0.01; ***p < 0.001.

  3. eCPMV inhalation reduces formation of B16F10 metastatic-like lung tumours.
    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. *p < 0.05; **p < 0.01; ***p < 0.001.

  4. eCPMV treatment efficacy in B16F10 lung model is immune-mediated.
    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. *p < 0.05; **p < 0.01; ***p < 0.001.

  5. eCPMV immunotherapy is successful in metastatic breast, colon, and ovarian carcinoma models.
    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 used the log-rank Mantel-Cox test for survival analysis and flank tumour growth curves were analysed using two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

  6. eCPMV induces systemic, durable antitumour immunity.
    Figure 6: eCPMV induces systemic, durable antitumour 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 used the log-rank Mantel-Cox test for survival analysis and flank tumour growth curves were analysed using two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

References

  1. Pardoll, D. M. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252264 (2012).
  2. Kershaw, M. H., Westwood, J. A. & Darcy, P. K. Gene-engineered T cells for cancer therapy. Nat. Rev. Cancer 13, 525541 (2013).
  3. Ali, O. A. et al. Identification of immune factors regulating antitumor immunity using polymeric vaccines with multiple adjuvants. Cancer Res. 74, 16701681 (2014).
  4. Callahan, M. K., Postow, M. A. & Wolchok, J. D. CTLA-4 and PD-1 pathway blockade: combinations in the clinic. Front. Oncol. 4, (2015).
  5. Winograd, R. et al. Induction of T cell immunity overcomes complete resistance to PD-1 and CTLA-4 blockade and improves survival in pancreatic carcinoma. Cancer Immunol. Res. 3, 399411 (2015).
  6. Robert, C. et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N. Engl. J. Med. 364, 25172526 (2011).
  7. Andtbacka, R. H. I. et al. Talimogene laherparepvec improves durable response rate in patients with advanced melanoma. J. Clin. Oncol. 33, 27802788 (2015).
  8. Corrales, L. et al. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell Rep. 11, 10181030 (2015).
  9. Sheen, M. R., Lizotte, P. H., Toraya-Brown, S. & Fiering, S. Stimulating antitumor immunity with nanoparticles. WIREs Nanomed. Nanobiotechnol. 6, 496505 (2014).
  10. Halperin, S. A. et al. Comparison of safety and immunogenicity of two doses of investigational hepatitis B virus surface antigen co-administered with an immunostimulatory phosphorothioate oligodeoxyribonucleotide and three doses of a licensed hepatitis B vaccine in healthy adults 18–55 years of age. Vaccine 30, 25562563 (2012).
  11. Huber, B. et al. A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types. PLoS ONE 10, e0120152 (2015).
  12. Rynda-Apple, A., Patterson, D. P. & Douglas, T. Virus-like particles as antigenic nanomaterials for inducing protective immune responses in the lung. Nanomed. 9, 18571868 (2014).
  13. Rynda-Apple, A. et al. Virus-like particle-induced protection against MRSA pneumonia is dependent on IL-13 and enhancement of phagocyte function. Am. J. Pathol. 181, 196210 (2012).
  14. Wiley, J. A. et al. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses. PLoS ONE 4, e7142 (2009).
  15. Patterson, D. P., Rynda-Apple, A., Harmsen, A. L., Harmsen, A. G. & Douglas, T. Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza. ACS Nano 7, 30363044 (2013).
  16. Richert, L. E. et al. CD11c+ cells primed with unrelated antigens facilitate an accelerated immune response to influenza virus in mice. Eur. J. Immunol. 44, 397408 (2014).
  17. Saunders, K., Sainsbury, F. & Lomonossoff, G. P. Efficient generation of cowpea mosaicvirus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants. Virology 393, 329337 (2009).
  18. Aljabali, A. A. A., Shukla, S., Lomonossoff, G. P., Steinmetz, N. F. & Evans, D. J. CPMV-DOX delivers. Mol. Pharm. 10, 310 (2013).
  19. Yildiz, I., Lee, K. L., Chen, K., Shukla, S. & Steinmetz, N. F. Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: Cargo-loading and delivery. J. Controlled Release 172, 568578 (2013).
  20. Costantini, C. et al. Neutrophil activation and survival are modulated by interaction with NK cells. Int. Immunol. 22, 827-838 (2010).
  21. Gabrilovich, D. I., Ostrand-Rosenberg, S. & Bronte, V. Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 12, 253268 (2012).
  22. Fridlender, Z. G. et al. Polarization of tumor-associated neutrophil (TAN) phenotype by TGF-β: ‘N1’ versus ‘N2’ TAN. Cancer Cell 16, 183194 (2009).
  23. Mantovani, A., Cassatella, M. A., Costantini, C. & Jaillon, S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat. Rev. Immunol. 11, 519531 (2011).
  24. Zhu, M.-L., Nagavalli, A. & Su, M. A. Aire deficiency promotes TRP-1–specific immune rejection of melanoma. Cancer Res. 73, 21042116 (2013).
  25. Conejo-Garcia, J. R. et al. Tumor-infiltrating dendritic cell precursors recruited by a β-defensin contribute to vasculogenesis under the influence of Vegf-A. Nature Med. 10, 950958 (2004).
  26. Lebel, M.-È. et al. Nanoparticle adjuvant sensing by TLR7 enhances CD8+ T cell–mediated protection from listeria monocytogenes infection. J. Immunol. 192, 10711078 (2014).
  27. Link, A. et al. Innate immunity mediates follicular transport of particulate but not soluble protein antigen. J. Immunol. 188, 37243733 (2012).
  28. Wu, G. J. & Bruening, G. Two proteins from cowpea mosaic virus. Virology 46, 596612 (1971).
  29. Steinmetz, N. F., Cho, C.-F., Ablack, A., Lewis, J. D. & Manchester, M. Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells. Nanomed. 6, 351364 (2011).
  30. Satelli, A. & Li, S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell. Mol. Life Sci. 68, 30333046 (2011).
  31. Gonzalez, M. J., Plummer, E. M., Rae, C. S. & Manchester, M. Interaction of cowpea mosaic virus (CPMV) nanoparticles with antigen presenting cells in vitro and in vivo. PLoS ONE 4, e7981 (2009).
  32. Jablonska, J., Leschner, S., Westphal, K., Lienenklaus, S. & Weiss, S. Neutrophils responsive to endogenous IFN-β regulate tumor angiogenesis and growth in a mouse tumor model. J. Clin. Invest. 120, 11511164 (2010).
  33. Kuang, D.-M. et al. Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma. J. Hepatol. 54, 948955 (2011).
  34. Pekarek, L. A., Starr, B. A., Toledano, A. Y. & Schreiber, H. Inhibition of tumor growth by elimination of granulocytes. J. Exp. Med. 181, 435440 (1995).
  35. Wislez, M. et al. Hepatocyte growth factor production by neutrophils infiltrating bronchioloalveolar subtype pulmonary adenocarcinoma role in tumor progression and death. Cancer Res. 63, 14051412 (2003).
  36. Abdallah, D. S. A., Egan, C. E., Butcher, B. A. & Denkers, E. Y. Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation. Int. Immunol. 23, 317326 (2011).
  37. van Gisbergen, K. P. J. M., Sanchez-Hernandez, M., Geijtenbeek, T. B. H. & van Kooyk, Y. Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN. J. Exp. Med. 201, 12811292 (2005).
  38. Beauvillain, C. et al. Neutrophils efficiently cross-prime naive T cells in vivo. Blood 110, 29652973 (2007).
  39. Pelletier, M. et al. Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 115, 335343 (2010).
  40. Clancy-Thompson, E. et al. Peptide vaccination in montanide adjuvant induces and GM-CSF increases CXCR3 and cutaneous lymphocyte antigen expression by tumor antigen–specific CD8 T cells. Cancer Immunol. Res. 1, 332339 (2013).
  41. Baird, J. R. et al. Immune-mediated regression of established B16F10 melanoma by intratumoral injection of attenuated toxoplasma gondii protects against rechallenge. J. Immunol. 190, 469478 (2013).
  42. Caramori, G., Adcock, I. M., Di Stefano, A. & Chung, K. F. Cytokine inhibition in the treatment of COPD. Int. J. Chron. Obstruct. Pulmon. Dis. 9, 397412 (2014).
  43. Lizotte, P. H. et al. Attenuated Listeria monocytogenes reprograms M2-polarized tumor-associated macrophages in ovarian cancer leading to iNOS-mediated tumor cell lysis. Oncoimmunology 3, (2014).
  44. Baird, J. R. et al. Avirulent toxoplasma gondii generates therapeutic antitumor immunity by reversing immunosuppression in the ovarian cancer microenvironment. Cancer Res. 73, 38423851 (2013).
  45. Scarlett, U. K. et al. In situ stimulation of CD40 and toll-like receptor 3 transforms ovarian cancer–infiltrating dendritic cells from immunosuppressive to immunostimulatory cells. Cancer Res. 69, 73297337 (2009).
  46. Hart, K., Byrne, K., Molloy, M., Usherwood, E. & Berwin, B. IL-10 immunomodulation of myeloid cells regulates a murine model of ovarian cancer. T Cell Biol. 2, 29 (2011).
  47. Sainsbury, F. et al. Genetic engineering and characterisation of Cowpea mosaic virus empty virus-like particles. Methods Mol Biol Clifton NJ. 1108, 139153 (2014).
  48. Lizotte, P. H., et al. Attenuated Listeria monocytogenes reprograms M2-polarized tumour-associated macrophages in ovarian cancer leading to iNOS-mediated tumour cell lysis. Oncoimmunology 3, e28926 (2014)
  49. Pulaski, B. A. & Ostrand-Rosenberg, S. in Current Protocols in Immunology (eds Cooligan, J. E. et al.) 39:20.2:20.2.120.2.16 (John Wiley & Sons, 2001); http://onlinelibrary.wiley.com/doi/10.1002/0471142735.im2002s39/abstract.

Download references

Author information

Affiliations

  1. Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire 03756, USA

    • P. H. Lizotte,
    • M. R. Sheen,
    • J. Fields,
    • P. Rojanasopondist &
    • S. Fiering
  2. Biomedical Engineering, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA

    • A. M. Wen &
    • N. F. Steinmetz
  3. Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA

    • N. F. Steinmetz
  4. Department of Materials Science and Engineering, Case School of Engineering, Cleveland, Ohio 44106, USA

    • N. F. Steinmetz
  5. Department of Macromolecular Science and Engineering, Case School of Engineering, Cleveland, Ohio 44106, USA

    • N. F. Steinmetz
  6. Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA

    • N. F. Steinmetz
  7. Department of Genetics, The Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire 03756, USA

    • S. Fiering
  8. Norris Cotton Cancer Center, Lebanon, New Hampshire 03756, USA

    • S. Fiering

Contributions

P.H.L., N.F.S., and S.F. conceived and designed the experiments, and wrote the manuscript. P.H.L., A.M.W., P.R., M.R.S., and J.F. performed the experiments. M.R.S. was responsible for Supplementary Fig. 4. P.R. was responsible for Supplementary Fig. 6. A.M.W. was responsible for Supplementary Fig. 8. P.H.L. performed all other experiments and analysed the data. J.F. assisted with in vivo work. All authors commented on the manuscript.

Competing financial interests

The authors have a patent pending for the immunotherapeutic use of the eCPMV nanoparticle.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (760 KB)

    Supplementary information

Additional data