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

Abstract

BackgroundAlthough 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.MethodsBinding 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.ResultsWe 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.ConclusionsThis new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.

Effects ofPortulaca oleracea L.Polysaccharides on Phenotypic and Functional Maturation of Murine Bone Marrow Derived Dendritic Cells

“…The animals were treated according to the National Institute of Health Guide for the Care and Use of Laboratory Animals and further approval for their experimentation was obtained from the Animal Ethics Committee of the university. CD11c C DCs were generated from bone marrow cells of C57BL/6 mice as described (14), with minor modifications. Briefly, mice were humanely killed by cervical dislocation, and bone marrow cells were recovered and flushed from femurs using enriched RPMI1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 mg/mL penicillin, and 100 U/mL streptomycin on ice.…”

Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease

“…Differential proteomics also established Hsp70 as a common antigen of Helicobacter pylori (26). Its adjuvant-type capacity even proved useful in tumor vaccination strategies, fusing Hsp70 to mesothelin or the oncoprotein E7 from human papillomavirus 16 (27,28). In line with this, complexing potential antigens with a stress protein like Hsp70 or GroEL, belonging to the chaperonins in the chaperone family, provides potent immunogens for CD8 ϩ T-cell activation (29).…”

“…Hsp70 was established as a common antigen of Leishmania infantum (25) and Helicobacter pylori (26), and the Th2 response to Hsp70 of Salmonella typhi protects mice from lethal Salmonella infection (30). Tumor vaccination strategies for human papillomavirus 16 benefit from the adjuvant-type capacity of Hsp70 by fusing it to mesothelin or the oncoprotein E7 (27,28), and the complexing of potential antigens with Hsp70 or GroEL provides immunogens for CD8 ϩ T-cell activation (29).…”

Latest developments in our understanding of the pathogenesis of mesothelioma and the design of targeted therapies

“…A mesothelin-targeted interleukin-12 conjugate showed some promising preclinical efficacy, and represented the first reported immunocytokine for mesothelin-positive tumors [178]. Moreover, a mesothelin-targeted Mycobacterium tuberculosis heat shock protein 70 promoted T cells activity against mesothelin-expressing cancers [179]. …”