A Novel Immunomodulatory Hemocyanin from the Limpet Fissurella latimarginata Promotes Potent Anti-Tumor Activity in Melanoma

Abstract

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.

Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages

“…Lyophilized KLH (from M. crenulata) in PBS was purchased from Thermo Scientific (Waltham, MA) and Calbiochem (San Diego, CA). Deglycosylated CCH and FLH were produced by sodium periodate treatment using the procedure and controls in Arancibia et al (8). To ensure that the biological effects observed were due to the hemocyanin component, the endotoxin contents of the hemocyanin preparations were determined using a PyroGene recombinant factor C endotoxin detection assay kit (Lonza Group, Walkersville, MD).…”

“…However, the biodiversity of hemocyanins has prompted interest in developing new candidates with better biochemical and immunological properties because the supply depends on natural resources. Therefore, hemocyanins from Concholepas concholepas (CCH) (7) and Fissurella latimarginata (FLH) (8), among others (6), have been studied. CCH is used as a carrier (9) and experimental Ag (10), and it has been preclinically evaluated in a murine model of superficial bladder cancer and showed antitumor effects similar to KLH (11).…”

“…Additionally, CCH has been used as an adjuvant in dendritic cell (DC) immunotherapy of patients with prostate cancer, and it is considered a safe complement or alternative to KLH (12,13). In contrast, FLH has shown intrinsically greater immunogenic capabilities, exhibiting a more potent antitumor activity than CCH and KLH against melanoma in the B16F10 mouse melanoma model (8).…”

“…In contrast, the two subunits of CCH are intermingled in the molecule, forming heterodidecamers (7). FLH is composed of a single type of subunit that forms homodidecamers (8). Another significant feature of the hemocyanin structure is the presence of carbohydrates, as heterogeneous N-and O-glycosylation ramifications, with mannose being the common and most abundant oligosaccharide (25)(26)(27)(28)(29)(30).…”

“…Although the three hemocyanins present N-and O-linked oligosaccharides, there are differences in their arrangements. For instance, KLH and FLH present xylose and sialic acid, but CCH is devoid of these sugars (8).…”