Valerie R. Wiersma

Therapeutic potential of Galectin-9 in human disease

In recent years, an important role has emerged for the glycan‐binding protein Galectin‐9 (Gal‐9) in health and disease. In normal physiology, Gal‐9 seems to be a pivotal modulator of T‐cell immunity by inducing apoptosis in specific T‐cell subpopulations. Because these T‐cell populations are associated with autoimmunity, inflammatory disease, and graft rejection, it was postulated that application of exogenous Gal‐9 may limit pathogenic T‐cell activity. Indeed, treatment with recombinant Gal‐9 ameliorates disease activity in various preclinical models of autoimmunity and allograft graft rejection. In many solid cancers, the loss of Gal‐9 expression is closely associated with metastatic progression. In line with this observation, treatment with recombinant Gal‐9 prevents metastatic spread in various preclinical cancer models. In addition, various hematological malignancies are sensitive to apoptotic elimination by recombinant Gal‐9. Here, we review the biology and physiological role of this versatile lectin and discuss the therapeutic potential of Gal‐9 in various diseases, including autoimmunity, asthma, infection, and cancer.

Mechanisms of Translocation of ER Chaperones to the Cell Surface and Immunomodulatory Roles in Cancer and Autoimmunity.

Endoplasmic reticulum (ER) chaperones (e.g., calreticulin, heat shock proteins, and isomerases) perform a multitude of functions within the ER. However, many of these chaperones can translocate to the cytosol and eventually the surface of cells, particularly during ER stress induced by e.g., drugs, UV irradiation, and microbial stimuli. Once on the cell surface or in the extracellular space, the ER chaperones can take on immunogenic characteristics, as mostly described in the context of cancer, appearing as damage-associated molecular patterns recognized by the immune system. How ER chaperones relocate to the cell surface and interact with other intracellular proteins appears to influence whether a tumor cell is targeted for cell death. The relocation of ER proteins to the cell surface can be exploited to target cancer cells for elimination by immune mechanism. Here we evaluate the evidence for the different mechanisms of ER protein translocation and binding to the cell surface and how ER protein translocation can act as a signal for cancer cells to undergo killing by immunogenic cell death and other cell death pathways. The release of chaperones can also exacerbate underlying autoimmune conditions, such as rheumatoid arthritis and multiple sclerosis, and the immunomodulatory role of extracellular chaperones as potential cancer immunotherapies requires cautious monitoring, particularly in cancer patients with underlying autoimmune disease.

Cell surface delivery of TRAIL strongly augments the tumoricidal activity of T-cells

Purpose: Adoptive T-cell therapy generally fails to induce meaningful anticancer responses in patients with solid tumors. Here, we present a novel strategy designed to selectively enhance the tumoricidal activity of T cells by targeted delivery of TNF-related apoptosis-inducing ligand (TRAIL) to the T-cell surface. Experimental Design: We constructed two recombinant fusion proteins, anti-CD3:TRAIL and K12:TRAIL. Tumoricidal activity of T cells in the presence of these fusion proteins was assessed in solid tumor cell lines, primary patient-derived malignant cells, and in a murine xenograft model. Results: When added to T cells, K12:TRAIL and anti-CD3:TRAIL selectively bind to the T-cell surface antigens CD3 and CD7, respectively, leading to cell surface accretion of TRAIL. Subsequently, anti-CD3:TRAIL and K12:TRAIL increased the tumoricidal activity of T cells toward cancer cell lines and primary patient-derived malignant cells by more than 500-fold. Furthermore, T-cell surface delivery of TRAIL strongly inhibited tumor growth and increased survival time of xenografted mice more than 6-fold. Conclusions: Targeted delivery of TRAIL to cell surface antigens of T cells potently enhances the tumoricidal activity of T cells. This approach may be generally applicable to enhance the efficacy of adoptive T-cell therapy. Clin Cancer Res; 17(17); 5626–37. ©2011 AACR.

The ever-expanding immunomodulatory role of calreticulin in cancer immunity.

Calreticulin is a pleiotropic molecule that normally resides in the lumen of the endoplasmic reticulum (ER). Here, it has various functions, ranging from regulation of calcium homeostasis to ensuring proper protein folding. More recently, calreticulin gained special interest for its extracellular functions, where it has direct immunomodulatory activity. In this respect, calreticulin activates dendritic cells and macrophages. In addition, certain anti-cancer therapies induce the translocation of calreticulin from the ER to the cell surface of dying cancer cells, where calreticulin dictates the immunogenicity of these cells. Interestingly, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) also induces membrane calreticulin exposure on cancer cells. As shown here, calreticulin directly interacts with TRAIL and its receptor-signaling complex, as well as with other TNF family members. Of note, TRAIL is a well known immunomodulatory molecule, and is expressed on the surface of natural killer T-cells. Therefore, calreticulin may have an as yet unrecognized wide(r) impact on immunity, with the TNF-ligand family modulating virtually all aspects of the immune response.

Elevated serum CXCL16 is an independent predictor of poor survival in ovarian cancer and may reflect pro-metastatic ADAM protease activity.

Background:In certain cancers, expression of CXCL16 and its receptor CXCR6 associate with lymphocyte infiltration, possibly aiding anti-tumour immune response. In other cancers, CXCL16 and CXCR6 associate with pro-metastatic activity. In the current study, we aimed to characterise the role of CXCL16, sCXCL16, and CXCR6 in ovarian cancer (OC).Methods:CXCL16/CXCR6 expression was analysed on tissue microarray containing 306 OC patient samples. Pre-treatment serum sCXCL16 was determined in 118 patients using ELISA. In vitro, (primary) OC cells were treated with an ADAM-10/ADAM-17 inhibitor (TAPI-2) and an ADAM-10-specific inhibitor (GI254023x), whereupon CXCL16 levels were evaluated on the cell membrane (immunofluorescent analysis, western blots) and in culture supernatants (ELISA). In addition, cell migration was assessed using scratch assays.Results:sCXCL16 independently predicted for poor survival (hazard ratio=2.28, 95% confidence interval=1.29–4.02, P=0.005), whereas neither CXCL16 nor CXCR6 expression correlated with survival. Further, CXCL16/CXCR6 expression and serum sCXCL16 levels did not associate with lymphocyte infiltration. In vitro inhibition of both ADAM-17 and ADAM-10, but especially the latter, decreased CXCL16 membrane shedding and strongly reduced cell migration of A2780 and cultured primary OC-derived malignant cells.Conclusions:High serum sCXCL16 is a prognostic marker for poor survival of OC patients, possibly reflecting ADAM-10 and ADAM-17 pro-metastatic activity. Therefore, serum sCXCL16 levels may be a pseudomarker that identifies patients with highly metastatic tumours.

Galectin-9 activates and expands human T-helper 1 cells.

Galectin-9 (Gal-9) is known for induction of apoptosis in IFN-γ and IL-17 producing T-cells and amelioration of autoimmunity in murine models. On the other hand, Gal-9 induced IFN-γ positive T-cells in a sarcoma mouse model and in food allergy, suggesting that Gal-9 can have diametric effects on T-cell immunity. Here, we aimed to delineate the immunomodulatory effect of Gal-9 on human resting and ex vivo activated peripheral blood lymphocytes. Treatment of resting lymphocytes with low concentrations of Gal-9 (5–30 nM) induced apoptosis in ∼60% of T-cells after 1 day, but activated the surviving T-cells. These viable T-cells started to expand after 4 days with up to 6 cell divisions by day 7 and an associated shift from naïve towards central memory and IFN-γ producing phenotype. In the presence of T-cell activation signals (anti-CD3/IL-2) Gal-9 did not induce T-cell expansion, but shifted the CD4/CD8 balance towards a CD4-dominated T-cell response. Thus, Gal-9 activates resting T-cells in the absence of typical T-cell activating signals and promotes their transition to a TH1/C1 phenotype. In the presence of T-cell activating signals T-cell immunity is directed towards a CD4-driven response by Gal-9. Thus, Gal-9 may specifically enhance reactive immunological memory.

The Glycan-Binding Protein Galectin-9 Has Direct Apoptotic Activity toward Melanoma Cells

TO THE EDITOR In recent years, a regulatory role has emerged for the glycan-binding protein galectin-9 (Gal-9) in normal physiology and pathology (reviewed by Wiersma et al. (2011). In melanoma and other malignancies, the available data suggest that Gal-9 has a tumor-suppressor function, with loss of Gal-9 being closely associated with metastatic progression (Kageshita et al., 2002; Irie et al., 2005; Yamauchi et al., 2006; Liang et al., 2008). In particular, melanocytic nevi and primary melanoma lesions highly express Gal-9, whereas metastatic melanoma lesions have no or minimal expression of Gal-9 (Kageshita et al., 2002). Furthermore, ectopic expression of Gal-9 abrogates the formation of metastases by Gal-9-deficient B16F10 murine melanoma cells (Nobumoto et al., 2008). Similarly, treatment of Gal-9-deficient B16F10 cells with a recombinant form of Gal-9, designated Gal-9(0), strongly reduced metastasis formation (Nobumoto et al., 2008). This anti-metastatic activity of Gal-9(0) on B16F10 has been attributed mainly to inhibition of melanoma cell adhesion to endothelial cells and/or extracellular matrix components, such as collagen type I (collagen-I; Nobumoto et al., 2008). The data presented in the current letter suggest that within the 1-h time frame of adhesion-type assays, treatment with Gal-9(0) triggers early apoptotic cellular changes. In line with earlier findings, Gal-9(0) inhibits the adhesion of B16F10 and 7 human melanoma cell lines to collagen-I (Figure 1a). However, the morphology of Gal-9(0)-treated cells that had adhered to collagen-I-coated wells resembled that of dying cells (Figure 1b; illustrated for B16F10). Subsequent analysis of this melanoma cell line panel, as well as primary patient–derived malignant melanoma cells for the early apoptotic marker phosphatidyl serine (PS), revealed that treatment with Gal-9(0) induced ∼90% cell death within the time frame used in the adhesion assay (Figure 1c). Early apoptotic PS exposure was followed by apoptotic cell death within 24 h of treatment, as evidenced by loss of viability (Supplementary Figure S1a online), the presence of late apoptotic Annexin-V/PI double-positive cells (Supplementary Figure S1b and c online), and an increase in DNA fragmentation (Supplementary Figure S1d online). In primary human melanocytes, Gal-9(0) also triggered PS exposure, albeit to a lesser extent (Figure 1c; ∼55%). More importantly, the viability of these normal cells was not negatively affected after 24 h (Supplementary Figure S1a online). Thus, Gal-9(0) induces rapid apoptotic cell death in melanoma cells, but not in normal human melanocytes. PS exposure induced by Gal-9(0) was fully dependent on the glycan-binding specificity of Gal-9(0), as it was selectively blocked by the competitive Gal-9 inhibitor alpha-lactose but not by the irrelevant carbohydrate sucrose (Figure 1d). Sensitivity to Gal-9(0) did not or only weakly correlated with expression of endogenous Gal-9 (Supplementary Figure S2 online; r2=0.250). Time-course analysis in five of the human melanoma cell lines demonstrated that treatment with Gal-9(0) induced PS exposure in >60% of melanoma cells within 5 minutes of treatment (Figure 1e). Furthermore, the extent of PS exposure closely correlated with the inhibitory effect of Gal-9(0) on collagen I binding (Figure 1f, MM-RU; r2=0.693). Together, these data suggest that the biological effect of Gal-9(0) in adhesion assays is mediated, at least partly, through the induction of cell death. It is noteworthy that pan-caspase inhibition failed to block the anti-adhesive and apoptotic activity of Gal-9(0; Supplementary Figure S1e and f online). Thus, Gal-9(0)-mediated melanoma apoptosis does not require caspase activation, which is in line with e.g. reports on gal-1-mediated cell death of T cells (Hahn et al., 2004). Figure 1 Galectin-9 (Gal-9(0)) rapidly induces apoptosis in serum-free conditions. (a). Adhesion of B16F10 and a panel of human melanoma cell lines to collagen-I-coated wells is inhibited by recombinant Gal-9. In brief, 3 × 104 melanoma cells were added ... Adhesion assays are typically performed in serum-free conditions, whereas in normal physiological situations the presence of serum and/or plasma components may affect the biological activity of Gal-9. Indeed, it is well established that Gal-9 can interact with serum components (Cederfur et al., 2008). Therefore, the biological activity of Gal-9(0) was next evaluated in the presence of 10% fetal calf serum (FCS), the standard serum additive in cell death assays. The inclusion of 10% FCS in these apoptosis experiments completely abrogated the morphological changes in melanoma cells, with induction of PS exposure by Gal-9(0) being abrogated in six of the seven cell lines tested (Figure 2a). Similarly, PS exposure by Gal-9(0) was also strongly inhibited in the primary melanoma cells (Figure 2a). Indeed, FCS dose dependently inhibited PS exposure induced by Gal-9(0) (illustrated for cell line A2058 in Figure 2b) and blocked the binding of Gal-9(0) to A2058 cells (Figure 2c). When using dialysed FCS or heat-inactivated FCS, the activity of Gal-9(0) was still inhibited (Figure 2d). Thus, the inhibitory component present in FCS is not heat labile (i.e., complement factors) and is >10 kDa in size. Notably, FCS did not inhibit Gal-9(0) activity toward SK-MEL-28 cells (Figure 2a), which suggests that the inhibitory effect of FCS is not merely due to binding of a serum component to Gal-9(0). Possibly, a serum component may shield the receptor(s) of Gal-9 on most melanoma cells. On Sk-MEL-28 cells, Gal-9(0) may interact with an alternative receptor not subject to binding/inhibition by FCS. Figure 2 Cell death induction by galectin-9 (Gal-9(0)) is blocked by fetal calf serum (FCS) but not by human pooled plasma. (a) A panel of melanoma cells and primary patient–derived melanoma cells (n=1) were treated for 1 h with Gal-9(0) in standard ... Although FCS is the standard additive in in vitro cell death assays, a better approximation of physiological settings is the addition of human plasma. Importantly, 10% human pooled plasma did not abrogate Gal-9(0)-induced PS exposure in A2058 cells, with only a slight reduction in PS exposure compared with serum-free conditions (Figure 2d). Similar results were obtained in six melanoma cell lines and in primary patient–derived melanoma cells (Figure 2e). These experiments suggest that an important biological effect of Gal-9(0) on human melanoma cells is the induction of apoptosis. This biological effect of Gal-9(0) is masked by as yet unidentified components present in FCS, but is unmasked in serum-free conditions or when human plasma is used. Indeed, in standard FCS-containing culture conditions, apoptotic cell death of the melanoma cell line MM-RU was only detected after 72 h of treatment with Gal-9 (Kageshita et al., 2002). The use of FCS may similarly mask the biological activity of other gal family members. In this respect, gal-2, -3, -4, and -8 interact with various serum components (Cederfur et al., 2008). Notably, plasma levels of several of the gal family members are increased in malignancy (Barrow et al., 2011). The use of human pooled serum/plasma instead of FCS for in vitro biological assays with members of the Gal family therefore appears prudent. In conclusion, recombinant Gal-9 has a hitherto unrecognized cytotoxic effect toward human melanoma cells, which further highlights its potential therapeutic applicability for the treatment of human metastatic melanoma.

The epithelial polarity regulator LGALS9/galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRASmut) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRASmut CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAFmut). In KRASmut CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRASmut cancer cells. Thus, rLGALS9 has potent antitumor activity toward refractory KRASmut CRC cells that may be exploitable for therapeutic use.

C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity.

The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.

A CD47-blocking TRAIL fusion protein with dual pro-phagocytic and pro-apoptotic anticancer activity

The expedient removal of dying, damaged or altered cells by phagocytosis is essential for homeostasis. However, cancer cells can evade such phagocytic elimination by cell surfaceupregulation of phagocyte-inhibitory signals, such as CD47. CD47 is a prominent ‘don’t eat me’ signal that binds to signalregulatory protein alpha (SIRPa/SIRPA) expressed on phagocytes (Oldenborg et al, 2001). The CD47-SIRPa interaction triggers phosphorylation of the immunoreceptor tyrosinebased inhibition motif (ITIM) of SIRPa and thereby potently inhibits phagocyte activity. Both solid and haematological malignancies hijack this inhibitory pathway by overexpression of CD47 (Chao et al, 2010, 2011; Zhao et al, 2011; Willingham et al, 2012). Recent studies indicated that blocking of CD47-SIRPa interaction promotes phagocytic elimination of CD47 overexpressing tumour cells (Chao et al, 2010; Kim et al, 2012). For instance, treatment of human B-cell non-Hodgkin lymphomas (B-NHL)-engrafted mice with CD47-blocking monoclonal antibody (MAb) B6H12 reduced lymphoma burden, improved survival and inhibited extranodal dissemination (Chao et al, 2010, 2011). Further combination of this CD47blocking antibody with the therapeutic antibody rituximab (RTX; a chimeric anti-CD20 IgG1) triggered synergistic anticancer activity in vivo (Chao et al, 2010). In addition, inhibition of CD47-SIRPa interaction enhanced the killing of trastuzumab-opsonized breast cancer cells (Zhao et al, 2011). Thus, CD47-SIRPa blocking strategies can enhance the efficacy of anticancer antibodies. Phagocytosis induced by RTX was also enhanced by F(ab’) 2 fragments of MAb B6H12 (Chao et al, 2010). This finding opens up the possibility for design of immunotherapeutics that combine CD47 blockade with alternate effector moieties. Here, we explored this possibility by genetic fusion of a CD47-blocking antibody fragment (scFv) to the pro-apoptotic immune effector molecule TRAIL (tumour necrosis factor [TNF]-related apoptosis-inducing ligand). TRAIL is a death ligand of the TNF-ligand superfamily that has pronounced tumour-selective pro-apoptotic activity (reviewed in (Bremer et al, 2009)). In phase I clinical trials, TRAIL treatment triggered minimal toxicity and, when combined with RTX, produced clinical responses in B-NHL patients (Fox et al, 2010). This new fusion protein, designated anti-CD47:TRAIL, was designed to 1) block CD47-SIRPa interaction and hereby potentiate phagocytosis induced by RTX, and 2) concurrently trigger CD47-restricted apoptotic cell death in malignant B-cells. To assess the effect of anti-CD47:TRAIL on RTX-induced phagocytosis, we performed mixed culture experiments with B-NHL cells and granulocytes as phagocytic effector cells as they are one of the most prevalent population of professional phagocytes. To this end, 1,10 -dioctadecyl-3,3,30,30 –tetramethylindodicarbocyanine (DiD)-labelled CD20/CD47 B-NHL cells (Fig. S1A–B) were mixed with granulocytes and incubated in the presence of RTX, MAb B6H12 or anti-CD47:TRAIL and combinations thereof. Subsequently, phagocytosis was determined by flow cytometry (see Fig. S1C for gating strategy). Treatment with RTX induced rapid phagocytosis of CD20 B-NHL cells, whereas treatment with anti-CD47:TRAIL alone did not (Fig. 1A). However, cotreatment with RTX and anti-CD47:TRAIL significantly increased tumour cell phagocytosis compared to RTX alone (Fig. 1A, P < 0 05). These flow cytometry data were corroborated by microscopy data, which revealed prominent tumour cell engulfment by granulocytes upon co-treatment (Fig. 1B). The potentiating effect of anti-CD47:TRAIL on RTX-mediated phagocytosis was dose-dependent and apparent at low ng/ml concentrations of anti-CD47:TRAIL (Fig. 1C). Importantly, anti-CD47:TRAIL also enhanced phagocytic removal of primary patient–derived B-NHL cells (Fig. 1D). Of note, at these concentrations MAb B6H12 did not potentiate RTX-induced phagocytosis (Fig. 1A-D), which is in apparent contrast with a previous report in which MAb B6H12 did synergize RTX-mediated phagocytosis (Chao et al, 2010). However, in our experiments we used significantly lower concentrations of both RTX and MAb B6H12 (RTX; 2 5 lg/ml vs. 10 lg/ml, MAb B6H12; 250 ng/ml vs. 10 lg/ml, respectively). Further, we used granulocytes as phagocytic effector cells, whereas Chao et al (2010) used macrophages. Third, TRAIL forms a stable homotrimer in scFv:TRAIL proteins (Bremer et al, 2004). Hence, trivalent binding by anti-CD47:TRAIL may result in a significantly higher CD47 blocking capacity compared with the bivalent blocking capacity of MAb B6H12. Phagocytosis induction by RTX and anti-CD47:TRAIL was abrogated at 0°C, indicating that tumour cells were eliminated by active phagocytosis (Fig. 1E). Furthermore, co-treatment of CD20 Namalwa cells with RTX and anti-CD47:TRAIL did not enhance phagocytosis. Likewise, co-treatment of B-cell lines with anti-CD47:TRAIL and cetuximab (CTX; a chimeric anti-epidermal growth factor receptor IgG1) failed to enhance phagocytosis (Fig. 1F). Thus, anti-CD47:TRAIL selectively enhanced antibody-mediated phagocytosis of B-NHL cells by RTX in a target antigen-restricted manner. Previously, we and others demonstrated that scFv:TRAIL fusion proteins have target antigen-restricted pro-apoptotic activity towards cancer cells (reviewed in (Bremer et al, 2009)). In line with this, anti-CD47:TRAIL triggered apoptosis in CD47 B-cell lines and in 4 of 5 primary malignant B-NHL samples (Fig. 2A, 2B). Importantly, normal blood cells were Correspondence