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A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma.

  • Author(s): Yuan, Jianping
  • Kashiwagi, Satoshi
  • Reeves, Patrick
  • Nezivar, Jean
  • Yang, Yuan
  • Arrifin, Nadiah Hashim
  • Nguyen, Mai
  • Jean-Mary, Gilberte
  • Tong, Xiaoyun
  • Uppal, Paramjit
  • Korochkina, Svetlana
  • Forbes, Ben
  • Chen, Tao
  • Righi, Elda
  • Bronson, Roderick
  • Chen, Huabiao
  • Orsulic, Sandra
  • Brauns, Timothy
  • Leblanc, Pierre
  • Scholler, Nathalie
  • Dranoff, Glenn
  • Gelfand, Jeffrey
  • Poznansky, Mark C
  • et al.
Abstract

Background

Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer, their production and administration is costly and labor-intensive. We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN), which is overexpressed on ovarian cancer and mesothelioma cells, to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is a potent immune activator that stimulates monocytes and DCs, enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs.

Methods

Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (i.p.) treatments with experimental or control proteins post i.p. injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses, a time-matched study was performed. Splenocytes were stimulated with peptides, and IFNγ- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs.

Results

We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells, and induced maturation of bone marrow-derived DCs. Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs.

Conclusions

This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.

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