RESUMO
Conjugation to carrier proteins is a way to improve the immunogenicity of peptides. Such is the case for peptides mimicking carbohydrate tumor-associated antigens in cancer vaccine development. The most used protein for this purpose is the keyhole limpet hemocyanin (KLH) from Megathura crenulata. Its limited bioavailability has prompted interest in finding new candidates; nevertheless, it is not known whether other hemocyanins might be equally efficient as carrier of carbohydrate peptide mimotopes to promotes anti-tumor responses. Here, we evaluated the carrier and antitumor activity of novel hemocyanins with documented immunogenicity obtained from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH), coupled through sulfo-SMCC to P10, a mimetic peptide of GD2, the major ganglioside constituent of neuroectodermal tumors, and incorporating AddaVax as an adjuvant. The humoral immune responses of mice showed that CCH-P10 and FLH-P10 conjugates elicited specific IgM and IgG antibodies against P10 mimotope, similar to those obtained with KLH-P10, which was used as a positive control. The CCH-P10 and FLH-P10 antisera, exhibited cross-reactivity with murine and human melanoma cells, like anti-CCH and anti-FLH sera suggesting a cross-reaction of CCH and FLH glycosylations with carbohydrate epitopes on the tumor cell surfaces, similar to the KLH antisera. When mice were primed with each hemocyanin-P10 and challenged with melanoma cells, better antitumor effects were observed for FLH-P10 than for CCH-P10 and, as for KLH-P10, irrespective of conjugation. These data demonstrate that CCH and FLH are useful carriers of carbohydrate mimotopes; however, the best antitumor activity of FLH preparations, indicate that is a suitable candidate for further cancer vaccines research.
Assuntos
Antineoplásicos/farmacologia , Gangliosídeos/farmacologia , Hemocianinas/farmacologia , Melanoma/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Gangliosídeos/química , Gastrópodes/química , Hemocianinas/química , Imunoterapia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Hemocyanins, the huge oxygen-transporting glycoproteins of some mollusks, are used as immunomodulatory proteins with proven anti-cancer properties. The biodiversity of hemocyanins has promoted interest in identifying new anti-cancer candidates with improved immunological properties. Hemocyanins promote Th1 responses without known side effects, which make them ideal for long-term sustained treatment of cancer. In this study, we evaluated a novel hemocyanin from the limpet/gastropod Fissurella latimarginata (FLH). This protein has the typical hollow, cylindrical structure of other known hemocyanins, such as the keyhole limpet hemocyanin (KLH) and the Concholepas hemocyanin (CCH). FLH, like the KLH isoforms, is composed of a single type of polypeptide with exposed N- and O-linked oligosaccharides. However, its immunogenicity was significantly greater than that of KLH and CCH, as FLH induced a stronger humoral immune response and had more potent anti-tumor activity, delaying tumor growth and increasing the survival of mice challenged with B16F10 melanoma cells, in prophylactic and therapeutic settings. Additionally, FLH-treated mice demonstrated increased IFN-γ production and higher numbers of tumor-infiltrating CD4(+) lymphocytes. Furthermore, in vitro assays demonstrated that FLH, but not CCH or KLH, stimulated the rapid production of pro-inflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α) by dendritic cells, triggering a pro-inflammatory milieu that may explain its enhanced immunological activity. Moreover, this effect was abolished when deglycosylated FLH was used, suggesting that carbohydrates play a crucial role in the innate immune recognition of this protein. Altogether, our data demonstrate that FLH possesses increased anti-tumor activity in part because it activates a more potent innate immune response in comparison to other known hemocyanins. In conclusion, FLH is a potential new marine adjuvant for immunization and possible cancer immunotherapy.