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

Our plain-English summary of A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma, by J. Yuan, S. Kashiwagi, P. Reeves, et al (Journal of Hematology & Oncology, 2014).

The study

• Authors: J. Yuan, S. Kashiwagi, P. Reeves, J. Nezivar, Y. Yang, N.H. Arrifin, M. Nguyen, G. Jean-Mary, X. Tong, P. Uppal, S. Korochkina, B. Forbes, T. Chen, E. Righi, R. Bronson, H. Chen, S. Orsulic, T. Brauns, P. Leblanc, N. Scholler, G. Dranoff, J. Gelfand, M.C. Poznansky
• Journal: Journal of Hematology & Oncology
• Published: 2014;7:15
• Cited by: 34 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This was a laboratory and animal study of a new immunotherapy protein, not a study of people or of asbestos exposure. The authors fused a single-chain antibody fragment (P4 scFv) that targets mesothelin to Mycobacterium tuberculosis heat shock protein 70 (MTBHsp70). In cell experiments, the fusion protein bound BR5FVB1 ovarian cancer cells and 40L mesothelioma cells, and binding was almost completely blocked by preincubation with recombinant human MSLN; binding to normal mouse peritoneal mesothelial cells was low and to blood leukocytes and splenocytes very low or undetectable. Mice bearing tumours in the abdomen received four intraperitoneal treatments of either the fusion protein at 2 micrograms per mouse, normal saline, or an equimolar mixture of MTBHsp70 plus P4 scFv. In the ovarian model (10 mice per group) median ascites-free survival was 47 days with the fusion protein versus 37.5 days with saline and 39 days with the unlinked mixture; in the mesothelioma model (20 mice per group) it was 28 versus 26 and 27 days. In the ovarian model only, depleting CD8+ cells meant the fusion protein no longer delayed onset of clinically evident ascites compared with saline (32.5 versus 31.5 days; p = 0.5938). The authors concluded their study provides preclinical evidence supporting this protein-based immunotherapy.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Preclinical laboratory and animal experiment: in vitro binding and dendritic-cell assays plus syngeneic mouse tumour models
• Who was studied: Immune-competent mice bearing intraperitoneal tumours: papillary ovarian cancer (BR5FVB1 cells) in FVB/NJ mice and mesothelioma (40L cells) in C57BL/6 mice; also cultured tumour cells, mouse bone marrow-derived dendritic cells, peritoneal mesothelial cells, blood leukocytes and splenocytes
• Exposure: Treatment with the scFvMTBHsp70 fusion protein (2 micrograms per mouse, four intraperitoneal doses), compared with normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv
• Main results:
  • In the ovarian cancer model (10 mice per group), median ascites-free survival was 47 days with scFvMTBHsp70 versus 37.5 days with saline and 39 days with the MTBHsp70 plus P4 scFv mixture; median overall survival was 51.5 days versus 43 days and 43 days
  • In the mesothelioma model (20 mice per group), median ascites-free survival was 28 days with the fusion protein versus 26 days with saline and 27 days with the mixture; median overall survival was 36 days versus 31 days with saline
  • Binding of the fusion protein to the MSLN-expressing tumour cells was blocked by recombinant MSLN, while MTBHsp70 binding did not differ significantly from background (p = 0.187 for BR5FVB1 cells; p = 0.086 for 40L cells)
  • In the ovarian model, CD8+ cell-depleted mice treated with the fusion protein had median ascites-free survival of 32.5 days versus 48 days in non-depleted treated mice; after depletion the fusion protein did not differ from saline (32.5 versus 31.5 days; p = 0.5938)
  • In naive mice given the same dosing schedule, microscopic examination of abdominal and intestinal peritoneum showed no inflammation or inflammatory cell infiltration in treated or control animals
  • Numbers of tumour-infiltrating CD8+ T cells did not differ significantly between treatment groups and no tumour-infiltrating Foxp3+ T cells were seen
• The authors' conclusion: The authors concluded that their study gives preclinical evidence for a protein-based immunotherapy that induces anti-tumour immune responses normally requiring dendritic cell-based approaches, and that continued exploration of the fusion protein is supported
• Limitations the authors state:
  • The authors noted that at the dosing schedule used, the tumour-specific T-cell responses did not lead to rejection of the established tumours
  • The authors described the fusion protein as potentially able to target peritoneal mesothelial cells, which they had to test separately for inflammation

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma, Ovarian cancer.

Medical terms in this study, explained in our Lexicon: Mesothelin, Ovarian Cancer, Mesothelioma, Ascites, Overall Survival, Immunotherapy.

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