Cancer Immunology, Immunotherapy

, Volume 57, Issue 4, pp 507–515 | Cite as

A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis

  • Susanna Lewēn
  • He Zhou
  • Huai-dong Hu
  • Tingmei Cheng
  • Dorothy Markowitz
  • Ralph A. Reisfeld
  • Rong Xiang
  • Yunping Luo
Original Article


Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor’s stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam, AroA) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-γ release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8+ T-cell response against Legumain+ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.


Legumain Minigene vaccine Tumor associated macrophages Anti-angiogenesis CTLs 



Tumor associated macrophages


Cytotoxic T lymphocytes


Major histocompatibility complex


Tumor microenvironment





We thank K. Cairns for editorial assistance. This work was supported by Grant 12RT-0002 from the California Tobacco-Related Disease Research Program (to R.A.R.), and E. Merck, Darmstadt-Lexigen Research Center, Billerica, MA Grant SFP1330 (to R.A.R.). Grants from The National Natural Science Foundation of China No.30672389 and No. 30572116 (to R.X.). This is The Scripps Research Institute’s manuscript number 17696-IMM.


  1. 1.
    Luo Y, Zhou H, Krueger J, Kaplan C, Lee SH, Dolman C, Markowitz D, Wu W, Liu C, Reisfeld RA, Xiang R (2006) Targeting tumor-associated macrophages as a novel strategy against breast cancer. J Clin Invest 116:2132PubMedCrossRefGoogle Scholar
  2. 2.
    Oosterling SJ, van der Bij GJ, Meijer GA, Tuk CW, van GE, van RN, Meijer S, van dS Jr, Beelen RH, van EM (2005) Macrophages direct tumor histology and clinical outcome in a colon cancer model. J Pathol 207:147Google Scholar
  3. 3.
    Liu C, Sun C, Huang H, Janda K, Edgington T (2003) Overexpression of legumain in tumors is significant for invasion/metastasis and a candidate enzymatic target for prodrug therapy. Cancer Res 63:2957PubMedGoogle Scholar
  4. 4.
    Murthy RV, Arbman G, Gao J, Roodman GD, Sun XF (2005) Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer. Clin Cancer Res 11:2293PubMedCrossRefGoogle Scholar
  5. 5.
    Zhou H, Luo Y, Mizutani M, Mizutani N, Xiang R, Reisfeld RA (2005) T-cell-mediated suppression of angiogenesis results in tumor protective immunity. Blood 106:2026PubMedCrossRefGoogle Scholar
  6. 6.
    Xiang R, Lode HN, Chao TH, Ruehlmann JM, Dolman CS, Rodriguez F, Whitton JL, Overwijk WW, Restifo NP, Reisfeld RA (2000) An autologous oral DNA vaccine protects against murine melanoma. Proc Natl Acad Sci USA 97:5492PubMedCrossRefGoogle Scholar
  7. 7.
    Luo Y, Zhou H, Mizutani M, Mizutani N, Reisfeld RA, Xiang R (2003) Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine. Proc Natl Acad Sci USA 100:8850PubMedCrossRefGoogle Scholar
  8. 8.
    Luo Y, Zhou H, Mizutani M, Mizutani N, Liu C, Xiang R, Reisfeld RA (2005) A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence. Cancer Res 65:3419PubMedCrossRefGoogle Scholar
  9. 9.
    Zhou H, Luo Y, Lo JF, Kaplan CD, Mizutani M, Mizutani N, Lee JD, Primus FJ, Becker JC, Xiang R, Reisfeld RA (2005) DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor. Proc Natl Acad Sci USA 102:10846PubMedCrossRefGoogle Scholar
  10. 10.
    Zhou H, Luo Y, Mizutani M, Mizutani N, Becker JC, Primus FJ, Xiang R, Reisfeld RA (2004) A novel transgenic mouse model for immunological evaluation of carcinoembryonic antigen-based DNA minigene vaccines. J Clin Invest 113:1792PubMedCrossRefGoogle Scholar
  11. 11.
    Niethammer AG, Xiang R, Becker JC, Wodrich H, Pertl U, Karsten G, Eliceiri BP, Reisfeld RA (2002) A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 8:1369PubMedCrossRefGoogle Scholar
  12. 12.
    Sinha P, Clements VK, Ostrand-Rosenberg S (2005) Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease. J Immunol 174:636PubMedGoogle Scholar
  13. 13.
    Stein M, Keshav S, Harris N, Gordon S (1992) Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med 176:287PubMedCrossRefGoogle Scholar
  14. 14.
    Porcheray F, Viaud S, Rimaniol AC, Leone C, Samah B, reuddre-Bosquet N, Dormont D, Gras G (2005) Macrophage activation switching: an asset for the resolution of inflammation. Clin Exp Immunol 142:481PubMedGoogle Scholar
  15. 15.
    Hicklin DJ, Marincola FM, Ferrone S (1999) HLA class I antigen downregulation in human cancers: T-cell immunotherapy revives an old story. Mol Med Today 5:178PubMedCrossRefGoogle Scholar
  16. 16.
    Cohen S, Regev A, Lavi S (1997) Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability. Oncogene 14:977PubMedCrossRefGoogle Scholar
  17. 17.
    Boehm T, Folkman J, Browder T, O’Reilly MS (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404PubMedCrossRefGoogle Scholar
  18. 18.
    Vitale, Rezzani MR, Rodella L, Zauli G, Grigolato P, Cadei M, Hicklin DJ, Ferrone S (1998). HLA class I antigen and transpoter associated with antigen processing (TAP1 and TAP2) down-regulation in high-grade primary breast carcinona lesions. Cancer Res 58:737Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Susanna Lewēn
    • 1
  • He Zhou
    • 1
  • Huai-dong Hu
    • 2
  • Tingmei Cheng
    • 2
  • Dorothy Markowitz
    • 1
  • Ralph A. Reisfeld
    • 1
  • Rong Xiang
    • 1
    • 2
  • Yunping Luo
    • 1
  1. 1.Department of ImmunologyThe Scripps Research InstituteLa JollaUSA
  2. 2.Key Laboratory of Molecular Biology for Infectious Disease, Ministry of Education, Institute for Viral Hepatitis, Second Affiliated HospitalChongqing Medical UniversityChongqingChina

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