Immunotherapeutic Potential of Mollusk Hemocyanins in Combination with Human Vaccine Adjuvants in Murine Models of Oral Cancer.

Juan José Mora Román,Paola Murgas,Aldo Venuti,Lilian Jara,Javiera Villar,Augusto Manubens,Jorge Ferreira,Francesca Paolini,Alvaro Lladser,María Inés Becker,Gianfranca Curzio,Miguel Del Campo

doi:10.1155/2019/7076942

Abstract

Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line cultured in vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.

Highlights

Hemocyanins, large respiratory glycoproteins found in certain mollusks, have a broad range of beneficial biomedical effects due to their potent Th1 adjuvant activity [1]
Fissurella latimarginata hemocyanin head and neck squamous cell carcinoma (HNSCC) (FLH), and keyhole limpet hemocyanin (KLH) was used as positive control. These data demonstrated that hemocyanins in combination with QS-21 induced robust humoral and cell-mediated immune responses compared to hemocyanins in combination with alum or AddaVax
QS-21 combined with hemocyanins showed the best results, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a Delayed-Type Hypersensitivity Reaction (DTH) reaction, confirming that QS-21 stimulates the polarization of the cytokine environment to Th1 response; only half the recommended dose of QS-21 was used in the mice, AddaVax and alum were used at the maximum recommended dose [25, 69]

Summary

Introduction

Hemocyanins, large respiratory glycoproteins found in certain mollusks, have a broad range of beneficial biomedical effects due to their potent Th1 adjuvant activity [1]. KLH has been applied in various in vitro and preclinical studies to determine its effectiveness against other cancers, such as Barrett’s adenocarcinoma [8]; pancreatic, breast, and prostate cancer [9]; and melanoma [10, 11]. Gastropod hemocyanins from Concholepas concholepas (CCH) [12], Fissurella latimarginata (FLH) [13], Haliotis tuberculata [14], Helix pomatia [15], and Rapana venosa [16, 17], which have been extensively characterized in terms of their biochemical properties and immunomodulatory/adjuvant effects, have emerged as potential candidates to complement or substitute for KLH [1, 18]

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