Liposomal gp100 vaccine combined with CpG ODN sensitizes established B16F10 melanoma tumors to anti PD-1 therapy

Document Type : Original Article


1 Department of Applied Cell Sciences, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran

2 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

3 Anatomical Sciences Research Center, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran

4 Department of Pathology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

5 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

6 Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

7 Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Program death 1 (PD-1)/ program death-ligand 1 (PD-L1) pathways, as the main inhibitory checkpoints, induce immunosuppression in the tumor microenvironment (TME). Despite the importance of inhibitor checkpoint receptor (ICR) blockers, their outcomes have been limited by the low immune response rate and induced acquired resistance. Pre-existing tumor-specific T cells is related to the improvement of their therapeutic efficacy. In the present study, we show that the combination of liposomal gp100 nanovaccine with anti PD-1 monoclonal antibody (mAb) potentiates the therapeutic effect in the melanoma model.
Materials and Methods: In this study, we first decorate the cationic liposome with gp10025-33 self-antigen and then characterize it. Mice bearing B16F10 melanoma tumors were vaccinated with different formulations of gp100 peptide (free or liposomal form) with or without CpG ODN adjuvant in combination with anti PD-1 mAb.
Results: Therapeutic combination of liposomal nanovaccine and CpG with anti PD-1 mAb, demonstrated the increased number of tumor infiltrated lymphocytes (TILs) in TME with the highest IFN-γ production and cytotoxic activity, which led to remarkable tumor regression.
Conclusion: Our results demonstrated the synergism between Lip-peptide+CpG nanovaccine and anti PD-1 regime, which improved the therapeutic efficacy of PD-1 checkpoint blocker in melanoma mice models.


1. Fan Q CZ, Wang C, Liu Z. Toward biomaterials for enhancing immune checkpoint blockade therapy. Adv Funct Mater 2018; 28:1802540.
2. Van der Burg SH, Arens R, Ossendorp F, van Hall T, Melief CJ. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat Rev Cancer 2016; 16:219-233.
3. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 2012;12:298-306.
4. Spranger S, Koblish HK, Horton B, Scherle PA, Newton R, Gajewski TF. 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 2014; 2:3.
5. Gajewski TF. The next hurdle in cancer immunotherapy: Overcoming the non-T-cell-inflamed tumor microenvironment. Semin Oncol 2015; 42:663-671.
6. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature. 2014; 515:568-571.
7. Chowdhury PS, Chamoto K, Honjo T. Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy. J Intern Med 2018; 283:110-120.
8. Byrne KT, Cote AL, Zhang P, Steinberg SM, Guo Y, Allie R, et al. Autoimmune melanocyte destruction is required for robust CD8+ memory T cell responses to mouse melanoma. J Clin Invest 2011; 121:1797-1809.
9. Dougan M DG. Immune therapy for cancer. Annu Rev Immunol 2009; 27:83–117.
10. Smith DM, Simon JK, Baker JR, Jr. Applications of nanotechnology for immunology. Nat Rev Immunol 2013; 13:592-605.
11. Korsholm KS, Andersen PL, Christensen D. Cationic liposomal vaccine adjuvants in animal challenge models: overview and current clinical status. Expert Rev Vaccines 2012; 11:561-577.
12. Xiang SD, Scholzen A, Minigo G, David C, Apostolopoulos V, Mottram PL, et al. Pathogen recognition and development of particulate vaccines: does size matter? Methods 2006; 40:1-9.
13. Oussoren C, Zuidema J, Crommelin DJ, Storm G. Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid compostion and lipid dose. Biochim Biophys Acta 1997; 1328:261-272.
14. Christensen D, Korsholm KS, Rosenkrands I, Lindenstrom T, Andersen P, Agger EM. Cationic liposomes as vaccine adjuvants. Expert Rev Vaccines 2007; 6:785-796.
15. Vangasseri DP, Cui Z, Chen W, Hokey DA, Falo LD, Jr., Huang L. Immunostimulation of dendritic cells by cationic liposomes. Mol Membr Biol 2006; 23:385-395.
16. Yan W, Chen W, Huang L. Mechanism of adjuvant activity of cationic liposome: phosphorylation of a MAP kinase, ERK and induction of chemokines. Mol Immunol 2007; 44:3672-3681.
17. Steinhagen F, Kinjo T, Bode C, Klinman DM. TLR-based immune adjuvants. Vaccine 2011; 29:3341-3355.
18. Khazanov E, Simberg D, Barenholz Y. Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice. J Gene Med 2006; 8:998-1007.
19. Whitmore MM, Li S, Falo L, Jr., Huang L. Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses. Cancer Immunol Immunother 2001; 50:503-514.
20. Kaczanowska S, Joseph AM, Davila E. TLR agonists: our best frenemy in cancer immunotherapy. J Leukoc Biol 2013; 93:847-863.
21. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004; 5:190-198.
22. Klinman DM, Currie D, Gursel I, Verthelyi D. Use of CpG oligodeoxynucleotides as immune adjuvants. Immunol Rev 2004; 199:201-216.
23. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20:709-760.
24. Puangpetch A, Anderson R, Huang YY, Sermswan RW, Chaicumpa W, Sirisinha S, et al. Cationic liposomes extend the immunostimulatory effect of CpG oligodeoxynucleotide against Burkholderia pseudomallei infection in BALB/c mice. Clin Vaccine Immunol 2012; 19:675-683.
25. Davila E, Kennedy R, Celis E. Generation of antitumor immunity by cytotoxic T lymphocyte epitope peptide vaccination, CpG-oligodeoxynucleotide adjuvant, and CTLA-4 blockade. Cancer Res 2003; 63:3281-3288.
26. Mangsbo SM, Sandin LC, Anger K, Korman AJ, Loskog A, Totterman TH. Enhanced tumor eradication by combining CTLA-4 or PD-1 blockade with CpG therapy. J Immunother 2010; 33:225-235.
27. Wang S, Campos J, Gallotta M, Gong M, Crain C, Naik E, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016; 113:E7240- E7249.
28. Gholizadeh Z, Tavakkol-Afshari J, Nikpoor AR, Jalali SA, Jaafari MR. Enhanced immune response induced by P5 HER2/neu-derived peptide-pulsed dendritic cells as a preventive cancer vaccine. J Cell Mol Med 2018; 22:558-567.
29. Pappalardo F, Pennisi M, Ricupito A, Topputo F, Bellone M. Induction of T-cell memory by a dendritic cell vaccine: a computational model. Bioinformatics 2014; 30:1884-1891.
30. Filion MC, Phillips NC. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta 1997; 1329:345-356.
31. Moynihan KD, Opel CF, Szeto GL, Tzeng A, Zhu EF, Engreitz JM, et al. Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses. Nat Med 2016; 22:1402-1410.
32. Fu J, Malm IJ, Kadayakkara DK, Levitsky H, Pardoll D, Kim YJ. Preclinical evidence that PD1 blockade cooperates with cancer vaccine TEGVAX to elicit regression of established tumors. Cancer Res 2014; 74:4042-4052.
33. Blake SJ, Ching AL, Kenna TJ, Galea R, Large J, Yagita H, et al. Blockade of PD-1/PD-L1 promotes adoptive T-cell immunotherapy in a tolerogenic environment. PloS one 2015; 10:e0119483.
34. Shindo Y, Yoshimura K, Kuramasu A, Watanabe Y, Ito H, Kondo T, et al. Combination immunotherapy with 4-1BB activation and PD-1 blockade enhances antitumor efficacy in a mouse model of subcutaneous tumor. Anticancer Res 2015; 35:129-136.
35. Danciu C, Oprean C, Coricovac DE, Andreea C, Cimpean A, Radeke H, et al. Behaviour of four different B16 murine melanoma cell sublines: C57BL/6J skin. Int J Exp Pathol 2015; 96:73-80.
36. Satterlee AB, Rojas JD, Dayton PA, Huang L. Enhancing Nanoparticle Accumulation and Retention in Desmoplastic Tumors via Vascular Disruption for Internal Radiation Therapy. Theranostics 2017; 7:253-269.
37. Kalli F, Machiorlatti R, Battaglia F, Parodi A, Conteduca G, Ferrera F, et al. Comparative analysis of cancer vaccine settings for the selection of an effective protocol in mice. J Transl Med 2013; 11:120.
38. Schluep T, Hwang J, Cheng J, Heidel JD, Bartlett DW, Hollister B, et al. Preclinical efficacy of the camptothecin-polymer conjugate IT-101 in multiple cancer models. Clin Cancer Res 2006; 12:1606-1614.
39. Prezado Y, Sarun S, Gil S, Deman P, Bouchet A, Le Duc G. Increase of lifespan for glioma-bearing rats by using minibeam radiation therapy. J Synchrotron Radiat 2012; 19:60-65.
40. Mishina H, Watanabe K, Tamaru S, Watanabe Y, Fujioka D, Takahashi S, et al. Lack of phospholipase A2 receptor increases susceptibility to cardiac rupture after myocardial infarction. Circ Res 2014; 114:493-504.
41. Razazan A, Behravan J, Arab A, Barati N, Arabi L, Gholizadeh Z, et al. Conjugated nanoliposome with the HER2/neu-derived peptide GP2 as an effective vaccine against breast cancer in mice xenograft model. PloS one 2017; 12:e0185099.
42. Vasievich EA, Chen W, Huang L. Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine. Cancer Immunol Immunother 2011; 60:629-638.
43. Zeng Q, Jiang H, Wang T, Zhang Z, Gong T, Sun X. Cationic micelle delivery of Trp2 peptide for efficient lymphatic draining and enhanced cytotoxic T-lymphocyte responses. J Control Release 2015; 200:1-12.
44. Li SY, Liu Y, Xu CF, Shen S, Sun R, Du XJ, et al. Restoring anti-tumor functions of T cells via nanoparticle-mediated immune checkpoint modulation. J Control Release 2016; 231:17-28.
45. Chen S, Lee LF, Fisher TS, Jessen B, Elliott M, Evering W, et al. Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector/memory CD8 T cells in a poorly immunogenic tumor model. Cancer Immunol Res 2015; 3:149-160.
46. Gide TN, Wilmott JS, Scolyer RA, Long GV. Primary and Acquired Resistance to Immune Checkpoint Inhibitors in Metastatic Melanoma. Clin Cancer Res 2018; 24:1260-1270.
47. McDermott DF, Atkins MB. PD-1 as a potential target in cancer therapy. Cancer Med 2013; 2:662-673.
48. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 2012; 24:207-212.
49. Mahoney KM, Rennert PD, Freeman GJ. Combination cancer immunotherapy and new immunomodulatory targets. Nat Rev Drug Discov 2015; 14:561-584.
50. Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature 2017; 541:321-330.
51. Chen W, Yan W, Huang L. A simple but effective cancer vaccine consisting of an antigen and a cationic lipid. Cancer Immunol Immunother 2008; 57:517-530.
52. Saremi SS, Shahryari M, Ghoorchian R, Eshaghian H, Jalali SA, Nikpoor AR, et al. The role of nanoliposome bilayer composition containing soluble leishmania antigen on maturation and activation of dendritic cells. Iran J Basic Med Sci 2018; 21:536-545.
53. Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine 2004; 22:1903-1913.
54. Henriksen-Lacey M, Christensen D, Bramwell VW, Lindenstrom T, Agger EM, Andersen P, et al. Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response. J Control Release 2010; 145:102-108.
55. Henriksen-Lacey M, Christensen D, Bramwell VW, Lindenstrom T, Agger EM, Andersen P, et al. Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3beta-[N-(N’,N’-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses. Mol Pharm 2011; 8:153-161.
56. Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother 2005; 54:721-728.
57. Gao J, Ochyl LJ, Yang E, Moon JJ. Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens. International journal of nanomedicine 2017; 12:1251-1264.
58. Shahum E, Therien HM. Liposomal adjuvanticity: effect of encapsulation and surface-linkage on antibody production and proliferative response. Int J Immunopharmacol1995; 17:9-20.
59. Shariat S, Badiee A, Amir Jalali S, Mansourian M, Alireza Mortazavi S, Reza Jaafari M. Preparation and characterization of different liposomal formulations containing P5 HER2/neu-derived peptide and evaluation of their immunological responses and antitumor effects. Iran J Basic Med Sci 2015; 18:506-513.
60. Chen W, Huang L. Induction of cytotoxic T-lymphocytes and antitumor activity by a liposomal lipopeptide vaccine. Mol Pharm 2008; 5:464-471.
61. Shariat S, Badiee A, Jalali SA, Mansourian M, Yazdani M, Mortazavi SA, et al. P5 HER2/neu-derived peptide conjugated to liposomes containing MPL adjuvant as an effective prophylactic vaccine formulation for breast cancer. Cancer Lett 2014; 355:54-60.
62. Rastakhiz S, Yazdani M, Shariat S, Arab A, Momtazi-Borojeni AA, Barati N, et al. Preparation of nanoliposomes linked to HER2/neu-derived (P5) peptide containing MPL adjuvant as vaccine against breast cancer. J Cell Biochem 2018.
63. Farzad N, Barati N, Momtazi-Borojeni AA, Yazdani M, Arab A, Razazan A, et al. P435 HER2/neu-derived peptide conjugated to liposomes containing DOPE as an effective prophylactic vaccine formulation for breast cancer. Artif Cells Nanomed Biotechnol 2019; 47:665-673.
64. Zamani P, Navashenaq JG, Nikpoor AR, Hatamipour M, Oskuee RK, Badiee A, et al. MPL nano-liposomal vaccine containing P5 HER2/neu-derived peptide pulsed PADRE as an effective vaccine in a mice TUBO model of breast cancer. J Control Release 2019; 303:223-236.
65. Irvine DJ, Swartz MA, Szeto GL. Engineering synthetic vaccines using cues from natural immunity. Nat Mater 2013; 12:978-990.
66. Oyewumi MO, Kumar A, Cui Z. Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses. Expert Rev Vaccines 2010; 9:1095-1107.
67. Joshi MD, Unger WJ, Storm G, van Kooyk Y, Mastrobattista E. Targeting tumor antigens to dendritic cells using particulate carriers. J Control Release 2012; 161:25-37.
68. Bauer M, Redecke V, Ellwart JW, Scherer B, Kremer JP, Wagner H, et al. Bacterial CpG-DNA triggers activation and maturation of human CD11c-, CD123+ dendritic cells. J Immunol 2001; 166:5000-5007.
69. Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002; 20:621-667.
70. Dresch C, Leverrier Y, Marvel J, Shortman K. Development of antigen cross-presentation capacity in dendritic cells. Trends Immunol 2012; 33:381-388.
71. Brazolot Millan CL, Weeratna R, Krieg AM, Siegrist CA, Davis HL. CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proc Natl Acad Sci U S A 1998; 95:15553-15558.
72. Jalali SA, Sankian M, Tavakkol-Afshari J, Jaafari MR. Induction of tumor-specific immunity by multi-epitope rat HER2/neu-derived peptides encapsulated in LPD Nanoparticles. Nanomedicine 2012; 8:692-701.
73. Ghaffari-Nazari H, Tavakkol-Afshari J, Jaafari MR, Tahaghoghi-Hajghorbani S, Masoumi E, Jalali SA. Improving multi-epitope long peptide vaccine potency by using a strategy that enhances CD4+ T help in BALB/c Mice. PloS one 2015; 10:e0142563.
74. Tahaghoghi-Hajghorbani S, Tavakkol-Afshari J, Jaafari MR, Ghaffari-Nazari H, Masoumi E, Jalali SA. Improved immunogenicity against a Her2/neu-Derived peptide by employment of a Pan HLA DR-Binding epitope and CpG in a BALB/c mice model. Anticancer Agents Med Chem 2017; 17:851-858.
75. Jeanbart L, Ballester M, de Titta A, Corthesy P, Romero P, Hubbell JA, et al. Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes. Cancer Immunol Res 2014; 2:436-447.
76. Xu Z, Ramishetti S, Tseng YC, Guo S, Wang Y, Huang L. Multifunctional nanoparticles co-delivering Trp2 peptide and CpG adjuvant induce potent cytotoxic T-lymphocyte response against melanoma and its lung metastasis. J Control Release 2013; 172:259-265.
77. Mansourian M, Badiee A, Jalali SA, Shariat S, Yazdani M, Amin M, et al. Effective induction of anti-tumor immunity using p5 HER-2/neu derived peptide encapsulated in fusogenic DOTAP cationic liposomes co-administrated with CpG-ODN. Immunol Lett 2014; 162:87-93.