Abstract
The GM3(Neu5Gc) ganglioside represents a tumor-specific antigen that is considered a promising target for cancer immunotherapy. We previously demonstrated that the humanized antibody 14F7hT, specific for this ganglioside, exhibited significant antitumor effects in preclinical hematological tumor models. As this antibody recognizes human tumor tissues from several origins, we addressed its potential effect on different tumor types. The use of cell lines for testing GM3(Neu5Gc)-targeting strategies, in particular for human malignancies, is complicated by the absence in humans of functional cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMAH), the enzyme required for Neu5Gc sialic acid biosynthesis. Quantitative flow cytometry revealed the absence of surface GM3(Neu5Gc) in several human but also mouse cell lines, in the last case due to low expression of the enzyme. Hypoxia-induced expression of this ganglioside on human SKOV3 cells was observed upon culture in Neu5Gc-containing medium without evidence for CMAH-independent biosynthesis. However, only transfection of the mouse Cmah gene into human SKOV3 and mouse 3LL cells induced a stable expression of GM3(Neu5Gc) on the cancer cell surface, resulting in effective models to evaluate the antitumor responses by 14F7hT in vitro and in vivo. This antibody exerted antibody-dependent cell-mediated cytotoxicity (ADCC) and in vivo antitumor effects on these Cmah-transfected non-hematological tumors from both mouse and human origin. These results contribute to validate GM3(Neu5Gc) as a relevant target for cancer immunotherapy and reinforces the value of 14F7hT as a novel anti-cancer drug.
Highlights
For successful cancer immunotherapy, the nature of the targeted tumor-associated antigen is of critical importance
While by flow cytometric analysis the surface expression of GM3(Neu5Gc) on P3X63 cells was confirmed by staining with 14F7hT, no binding was detectable in other mouse cell lines, including 3LL Lewis lung carcinoma, 4T1 mammary carcinoma, B16-F10 melanoma, and ID8/MOSEC ovarian cancer cell lines (Fig. 1A)
Neu5Gc-sialoconjugates are found only in trace amounts in tissues[8], due to the inactivity of the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMAH) enzyme resulting from an exon deletion in the Cmah gene[4,5]
Summary
The nature of the targeted tumor-associated antigen is of critical importance. Given its tumor-associated expression, GM3(Neu5Gc)-targeting antibodies have been developed[3,20], including the mouse 14F7 monoclonal antibody[21], and its humanized variant 14F7hT22, for which promising antitumor effects were demonstrated using in vitro and in vivo models[22,23]. While hypoxia was shown to increase the uptake and metabolic incorporation of Neu5Gc from culture medium by the upregulation of the sialic acid transporter, sialin[25], in a recent article it was hypothesized that the enhanced GM3(Neu5Gc) expression under hypoxic conditions might be linked to CMAH-independent, alternate biosynthetic pathways in human cancer cells[12]. In line with the human-specific genetic inactivation of CMAH and in contradiction with previous reports[26,31,32,33], no GM3(Neu5Gc) surface expression was detected in human cell lines
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