Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators

Abstract

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: NP and man-NP are potential delivery systems for vaccination.
Fig. 2: NP and man-NP vaccines induce splenocyte activation and ex vivo cytotoxicity against melanoma cells.
Fig. 3: Prophylactic nanovaccines have a synergistic effect with PD-1 blockade and OX40 activation, which restricts melanoma growth and prolongs survival.
Fig. 4: Low CD8+:Treg ratio and high infiltration of MDSCs (CD11b+Gr-1+MDSC) compromise the therapeutic efficacy of the combination of mannosylated nanovaccines with αPD-1/αOX40.
Fig. 5: Trivalent combination of mannosylated nanovaccines with ibrutinib and αPD-1/αOX40 strongly restricts melanoma growth, which leads to long-term survival.
Fig. 6: Proposed model for the trivalent therapeutic strategy that combines mannosylated nanovaccines with ibrutinib and αPD-1/αOX40.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. Additional data and source files are available from the corresponding authors upon reasonable request.

References

  1. 1.

    Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. New Engl. J. Med. 366, 2443–2454 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Hodi, F. S. et al. Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711–723 (2010).

    CAS  Article  Google Scholar 

  3. 3.

    Gramaglia, I. et al. The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion. J. Immunol. 165, 3043–3050 (2000).

    CAS  Article  Google Scholar 

  4. 4.

    Arch, R. H. & Thompson, C. B. 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kappaB. Mol. Cell Biol. 18, 558–565 (1998).

    CAS  Article  Google Scholar 

  5. 5.

    Aspeslagh, S. et al. Rationale for anti-OX40 cancer immunotherapy. Eur. J. Cancer 52, 50–66 (2016).

    CAS  Article  Google Scholar 

  6. 6.

    Sarff, M. et al. OX40 (CD134) expression in sentinel lymph nodes correlates with prognostic features of primary melanomas. Am. J. Surg. 195, 621–625 (2008).

    CAS  Article  Google Scholar 

  7. 7.

    Vetto, J. T. et al. Presence of the T-cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers. Am. J. Surg. 174, 258–265 (1997).

    CAS  Article  Google Scholar 

  8. 8.

    Spranger, S. et al. Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8+ T cells directly within the tumor microenvironment. J. Immunother. Cancer 2, 3 (2014).

    Article  Google Scholar 

  9. 9.

    Gajewski, T. F. The next hurdle in cancer immunotherapy: overcoming the non-T-cell-inflamed tumor microenvironment. Semin. Oncol. 42, 663–671 (2015).

    Article  Google Scholar 

  10. 10.

    Minn, A. J. & Wherry, E. J. Combination cancer therapies with immune checkpoint blockade: convergence on interferon signaling. Cell 165, 272–275 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Tumeh, P. C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    van Kooyk, Y. C-type lectins on dendritic cells: key modulators for the induction of immune responses. Biochem Soc. Trans. 36, 1478–1481 (2008).

    Article  Google Scholar 

  13. 13.

    Kodumudi, K. N., Weber, A., Sarnaik, A. A. & Pilon-Thomas, S. Blockade of myeloid-derived suppressor cells after induction of lymphopenia improves adoptive T cell therapy in a murine model of melanoma. J. Immunol. 189, 5147–5154 (2012).

    CAS  Article  Google Scholar 

  14. 14.

    Meyer, C. et al. Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab. Cancer Immunol. Immunother. 63, 247–257 (2014).

    CAS  Article  Google Scholar 

  15. 15.

    Stiff, A. et al. Myeloid-derived suppressor cells express Bruton’s tyrosine kinase and can be depleted in tumor-bearing hosts by ibrutinib treatment. Cancer Res. 76, 2125–2136 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Natarajan, G. et al. A Tec kinase BTK inhibitor ibrutinib promotes maturation and activation of dendritic cells. Oncoimmunology 5, e115159 (2016).

    Google Scholar 

  17. 17.

    Alonso-Sande, M. et al. Development of PLGA–mannosamine nanoparticles as oral protein carriers. Biomacromolecules 14, 4046–4052 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Silva, J. M. et al. In vivo delivery of peptides and Toll-like receptor ligands by mannose-functionalized polymeric nanoparticles induces prophylactic and therapeutic anti-tumor immune responses in a melanoma model. J. Control Release 198, 91–103 (2015).

    CAS  Article  Google Scholar 

  19. 19.

    Wang, X., Ramstrom, O. & Yan, M. Dynamic light scattering as an efficient tool to study glyconanoparticle–lectin interactions. Analyst 136, 4174–4178 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    De Koker, S. et al. Engineering polymer hydrogel nanoparticles for lymph node-targeted delivery. Angew. Chem. Int. Ed. 55, 1334–1339 (2016).

    Article  Google Scholar 

  21. 21.

    Azzi, J. et al. Targeted delivery of immunomodulators to lymph nodes. Cell Rep. 15, 1202–1213 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Seliger, B., Ruiz-Cabello, F. & Garrido, F. IFN inducibility of major histocompatibility antigens in tumors. Adv. Cancer Res. 101, 249–276 (2008).

    CAS  Article  Google Scholar 

  23. 23.

    Dranoff, G. et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc. Natl Acad. Sci. USA 90, 3539–3543 (1993).

    CAS  Article  Google Scholar 

  24. 24.

    Kowalczyk, D. W. et al. Vaccine-induced CD8+ T cells eliminate tumors by a two-staged attack. Cancer Gene Ther. 10, 870–878 (2003).

    CAS  Article  Google Scholar 

  25. 25.

    Pasare, C. & Medzhitov, R. Toll-like receptors: balancing host resistance with immune tolerance. Curr. Opin. Immunol. 15, 677–682 (2003).

    CAS  Article  Google Scholar 

  26. 26.

    Scheller, J., Chalaris, A., Schmidt-Arras, D. & Rose-John, S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim. Biophys. Acta 1813, 878–888 (2011).

    CAS  Article  Google Scholar 

  27. 27.

    Fridlender, Z. G. et al. CCL2 blockade augments cancer immunotherapy. Cancer Res. 70, 109–118 (2010).

    CAS  Article  Google Scholar 

  28. 28.

    Tsui, P. et al. Generation, characterization and biological activity of CCL2 (MCP-1/JE) and CCL12 (MCP-5) specific antibodies. Hum. Antibodies 16, 117–125 (2007).

    CAS  Article  Google Scholar 

  29. 29.

    Phan, G. Q. et al. Immunization of patients with metastatic melanoma using both class I- and class II-restricted peptides from melanoma-associated antigens. J. Immunother. 26, 349–356 (2003).

    CAS  Article  Google Scholar 

  30. 30.

    Slingluff, C. L. Jr. et al. A randomized phase II trial of multiepitope vaccination with melanoma peptides for cytotoxic T cells and helper T cells for patients with metastatic melanoma (E1602). Clin. Cancer Res. 19, 4228–4238 (2013).

    CAS  Article  Google Scholar 

  31. 31.

    Shedlock, D. J. & Shen, H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300, 337–339 (2003).

    CAS  Article  Google Scholar 

  32. 32.

    Gabrilovich, D. I. & Nagaraj, S. Myeloid-derived suppressor cells as regulators of the immune system. Nat. Rev. Immunol. 9, 162–174 (2009).

    CAS  Article  Google Scholar 

  33. 33.

    Nagaraj, S., Schrum, A. G., Cho, H. I., Celis, E. & Gabrilovich, D. I. Mechanism of T cell tolerance induced by myeloid-derived suppressor cells. J. Immunol. 184, 3106–3116 (2010).

    CAS  Article  Google Scholar 

  34. 34.

    Sagiv-Barfi, I. et al. Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK. Proc. Natl Acad. Sci. USA 112, E966–E972 (2015).

    CAS  Article  Google Scholar 

  35. 35.

    Swart, M., Verbrugge, I. & Beltman, J. B. Combination approaches with immune-checkpoint blockade in cancer therapy. Front. Oncol. 6, 233 (2016).

    Article  Google Scholar 

  36. 36.

    Guo, Z. et al. PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer. PLoS ONE 9, e89350 (2014).

    Article  Google Scholar 

  37. 37.

    Woods, D. M., Ramakrishnan, R., Sodré, A. L., Berglund, A. & Weber, J. Abstract A067: PD-1 blockade enhances OX40 expression on regulatory T-cells and decreases suppressive function through induction of phospho-STAT3 signaling. Cancer Immunol. Res. 4, A067–A067 (2016).

    Google Scholar 

  38. 38.

    Zhu, Q. et al. Using 3 TLR ligands as a combination adjuvant induces qualitative changes in T cell responses needed for antiviral protection in mice. J. Clin. Invest. 120, 607–616 (2010).

    CAS  Article  Google Scholar 

  39. 39.

    Chen, L. & Flies, D. B. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat. Rev. Immunol. 13, 227–242 (2013).

    Article  Google Scholar 

  40. 40.

    Hanahan, D. & Coussens, L. M. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21, 309–322 (2012).

    CAS  Article  Google Scholar 

  41. 41.

    Burkholder, B. et al. Tumor-induced perturbations of cytokines and immune cell networks. Biochim. Biophys. Acta 1845, 182–201 (2014).

    CAS  Google Scholar 

  42. 42.

    Fang, H. et al. TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells. Cell. Mol. Immunol. 11, 150–159 (2013).

    Article  Google Scholar 

  43. 43.

    Beyersdorf, N., Kerkau, T. & Hunig, T. CD28 co-stimulation in T-cell homeostasis: a recent perspective. Immunotargets Ther. 4, 111–122 (2015).

    Google Scholar 

  44. 44.

    Sagiv-Barfi, I. et al. Eradication of spontaneous malignancy by local immunotherapy. Sci. Transl. Med. 10, eaan4488 (2018).

    Article  Google Scholar 

  45. 45.

    Maher, E. A., Mietz, J., Arteaga, C. L., DePinho, R. A. & Mohla, S. Brain metastasis: opportunities in basic and translational research. Cancer Res. 69, 6015–6020 (2009).

    CAS  Article  Google Scholar 

  46. 46.

    Santarelli, J. G., Sarkissian, V., Hou, L. C., Veeravagu, A. & Tse, V. Molecular events of brain metastasis. Neurosurg. Focus 22, E1 (2007).

    Article  Google Scholar 

  47. 47.

    van den Eertwegh, A. J. et al. Combined immunotherapy with granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells and ipilimumab in patients with metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial. Lancet Oncol. 13, 509–517 (2012).

    Article  Google Scholar 

  48. 48.

    Hodi, F. S. et al. Ipilimumab plus sargramostim vs ipilimumab alone for treatment of metastatic melanoma: a randomized clinical trial. J. Am. Med. Assoc. 312, 1744–1753 (2014).

    CAS  Article  Google Scholar 

  49. 49.

    Kaiser, A. D. et al. Towards a commercial process for the manufacture of genetically modified T cells for therapy. Cancer Gene Ther. 22, 72–78 (2015).

    CAS  Article  Google Scholar 

  50. 50.

    Wilson, D. S. et al. Antigens reversibly conjugated to a polymeric glyco-adjuvant induce protective humoral and cellular immunity. Nat. Mater. 18, 175–185 (2019).

    CAS  Article  Google Scholar 

  51. 51.

    Schwartz, H. et al. Incipient melanoma brain metastases instigate astrogliosis and neuroinflammation. Cancer Res. 76, 4359–4371 (2016).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The MultiNano@MBM project was supported by The Israeli Ministry of Health and The Fundação para a Ciência e Tecnologia-Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES) under the framework of EuroNanoMed-II (ENMed/0051/2016 to H.F.F., R.S.-F. and S.J.). R.S.-F. thanks the European Research Council (ERC) Consolidator Grant Agreement no. [617445]-PolyDorm and ERC Advanced Grant Agreement no. [835227] - 3DBrainStrom, The Israel Science Foundation (Grant nos. 918/14 and 1969/18), The Melanoma Research Alliance (the Saban Family Foundation–MRA Team Science Award to R.S.-F. and N.E., and Established Investigator Award to R.S.-F.) and the Israel Cancer Research Fund (ICRF). J.C., A.I.M., C.P., E.Z. and L.I.F.M. are supported by the FCT-MCTES (Fellowships SFRH/BD/87150/2012, PD/BD/113959/2015, SFRH/BD/87591/2012, SFRH/BD/78480/2011 and SFRH/BPD/94111/2013, respectively). This project has received funding from European Structural & Investment Funds through the COMPETE Programme and from National Funds through FCT under the Programme grant SAICTPAC/0019/2015 (H.F.F.). We thank E. Haimov and B. Redko from the Blavatnik Center for Drug Discovery at the Tel Aviv University for their professional and technical assistance with the peptide synthesis.

Author information

Affiliations

Authors

Contributions

J.C. and A.S. synthesized the nanovaccines and performed the in vitro and the animal studies; J.C. synthesized the man-PLGA polymers and carried out the physicochemical characterization of the nanovaccines; R.K. helped with the nanovaccine formulation; A.S., S.P. and R.K. performed the ELISPOT experiments; C.P. and A.I.M. analysed the flow cytometry experiments; E.Y. and S.P. performed the immunohistochemistry experiments; J.C. and A.I.M. carried out the tetramer assay; L.I.F.M. and E.Z. helped with the animal experiments; A.S.V. performed the AFM experiments; H.D. and N.E. contributed with the Ret cells; P.M.P.G. advised on the polymer synthesis; S.J. critically advised and contributed in interpreting the results. J.C., A.S., R.S.-F. and H.F.F. conceived and designed the experiments, analysed the data and wrote and revised the manuscript. R.S.-F. and H.F.F. were in charge of the overall direction and planning of this study, and all the authors commented on the manuscript.

Corresponding authors

Correspondence to Ronit Satchi-Fainaro or Helena F. Florindo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nature Nanotechnology thanks Walter Storkus and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figs. 1–19, Supplementary Tables 1–5 and Supplementary refs.

Reporting summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Conniot, J., Scomparin, A., Peres, C. et al. Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators. Nat. Nanotechnol. 14, 891–901 (2019). https://doi.org/10.1038/s41565-019-0512-0

Download citation

Further reading

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research