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Glioblastoma multiforme (GBM) is the most dramatic primary brain cancer with a very poor prognosis because of inevitable disease recurrence. The median overall survival is less than 1 year after diagnosis. Cancer stem cells have recently been disclosed in GBM. GBM stem-like cells (GSCs) exhibit resistance to radio/chemotherapeutic treatments and are therefore considered to play an important role in disease recurrence. GSCs are thus appealing targets for new treatments for GBM patients. In this study, we show that GBM cells with stem cell characteristics are resistant to lysis mediated by resting natural killer (NK) cells because of the expression of MHC class I molecules. However, GSCs are killed by lectin-activated NK cells. Furthermore, in experiments using the therapeutic antibody CetuximAb, we show that GSCs are sensitive to antibody-mediated cytotoxicity. We confirm the sensitivity of GSC to cytotoxicity carried out by IL2-activated NK cells and tumor-specific T cells. More importantly, we show that GSCs are more sensitive to NK and T cell-mediated lysis relatively to their corresponding serum-cultured GBM cells obtained from the same initial tumor specimen. Altogether, these results demonstrate the sensitivity of GSC to immune cell cytotoxicity and, therefore, strongly suggest that GSCs are suitable target cells for immunotherapy of GBM patients.

PI-2301 is an amino acid copolymer acting as an immunomodulator for the treatment of autoimmune diseases. The present study evaluated the safety, pharmacokinetics (PK), and pharmacodynamics of PI-2301 in a single ascending dose, first-in-human study involving healthy, male adult volunteers. A total of 56 subjects were given a subcutaneous injection of PI-2301 ranging from 0.035 to 60 mg. The only consistent side effect was transient injection site reactions. We describe, for the first time, a pharmacokinetic assay to monitor amino acid copolymer concentration in human serum. PI-2301 was detected in the serum of subjects in the 10-, 30-, and 60-mg cohorts. Maximum serum concentration was achieved between 10 and 30 minutes postdosing with some compound detected 4 hours after dosing. PI-2301's lasting immunological properties were evident by an ex vivo recall assay showing T-cell proliferation and IL-13 production in subjects dosed with 1, 3, or 10 mg of PI-2301, up to 6 months after dosing. A transient increase in chemokine CXCL9 and CXCL10 plasma levels was seen in subjects dosed with 30 or 60 mg of PI-2301. These results are highly consistent with our preclinical findings and suggest that PI-2301 could facilitate the expansion of a favorable immune posture in patients with autoimmune disorders.

Siglecs are a family of up to 11 transmembrane sialic acid-binding immunoglobulin superfamily. They are mostly expressed on discrete cellular subsets of the immune system where they are thought to be involved in both adhesive and signalling functions. Each siglec has a distinct preference for both the type of sialic acid as well as its linkage to other sugars. As sialic acids are commonly found on glycoproteins and glycolipids at the cell surface, siglecs can potentially interact with these ligands both in cis (on the same cell) as well as in trans (between cells). Cis interactions with sialic acids can mask the sialic acid binding activity of siglecs and unmasking can occur after sialidase treatment or in some cases by cellular activation. We have focused on siglec-7 as a paradigm for a new group of siglecs related to CD33 that includes siglecs-5, 6, 7, 8, 9, 10 and 11. Siglec-7 is expressed on mature human natural killer (NK) cells and binds preferentially to glycans containing α2,8-linked disialic acid. Similar to all CD33-related siglecs, siglec-7 has two tyrosine-based motifs that are predicted to mediate inhibitory signalling functions. When expressed on NK cells, siglec-7 is masked by cis interactions with sialic acids, but it can be unmasked following sialidase treatment. P815 target cells were transfected with GD3 synthase to induce expression of GD3, a simple ganglioside containing α2,8-linked disialic acid. Expression of GD3 increased their resistance to cytotoxicity mediated by sialidase-treated NK cell killing but had little effect on killing mediated by non sialidase-treated cells. To investigate whether NK cells might express surface α2,8-linked disialic acids that could explain the masked status of siglec-7, we have analysed the binding of antibodies thought to bind specifically to this structure. By FACS analysis, we observed preferential binding to NK cells compared to other blood leucocytes including B cells, T cells and monocytes. Real time PCR has been used to investigate expression of all known α2,8-specific sialyltransferases by NK cells and other blood leucocytes. Taken together, our findings indicate that NK cells express relatively high levels of α2,8-linked disialic acids and that the binding specificity of siglec-7 has become adapted to bind this structure, presumably to regulate NK-cell activation.