Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.
Bottom Line: Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant.In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261).Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF.
The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.
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Mentions: In order to evaluate the absolute concentration of 2HG, magnetic resonance spectroscopy (MRS) was performed on mice before and after (on day 8 and 20) pGL261 or mIDH1-GL261 implantation. A preliminary optimization of 2HG detection was performed by acquiring sequences with two different Echo Time values: TE = 13 ms and TE = 50 ms for each examination session (not shown). A more accurate quantification was obtained using spectra with the shorter TE, as also previously described . In pre-injection MRS data, there was no evidence of 2HG in all the examined animals (Figure 2a and b, left panels; quantification in Figure 2c). The improved signal-to noise ratio and the use of quantitative standards allowed to obtain for the first time an absolute quantification of 2HG in mouse brain. On day 8 mIDH1-GL261 gliomas showed a significantly higher concentration of 2HG as compared to pGL261 gliomas (4–7 mM vs 2 mM, Figure 2c and a, b right panels). Interestingly an accumulation of 2HG higher than the background was also reported in a subgroup of human wild type IDH gliomas . Decreased concentration of 2HG was detected on day 20 (Figure 2c). No difference in the absolute concentration of the other metabolites investigated was observed (e.g. choline, lactate, N-acetyl-aspartate). The levels of 2HG were directly measured by LC-MS/MS in gliomas explanted on day 30, confirming the presence of millimolar levels of the metabolite, with higher expression in the mIDH1-GL261 (Figure 2d).Figure 2