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.

Targeted delivery of CD40L promotes restricted activation of antigen-presenting cells and induction of cancer cell death.

BACKGROUND: Stimulation of CD40 can augment anti-cancer T cell immune responses by triggering effective activation and maturation of antigen-presenting cells (APCs). Although CD40 agonists have clinical activity in humans, the associated systemic activation of the immune system triggers dose-limiting side-effects. METHODS: To increase the tumor selectivity of CD40 agonist-based therapies, we developed an approach in which soluble trimeric CD40L (sCD40L) is genetically fused to tumor targeting antibody fragments, yielding scFv:CD40L fusion proteins. We hypothesized that scFv:CD40L fusion proteins would have reduced CD40 agonist activity similar to sCD40L but will be converted to a highly agonistic membrane CD40L-like form of CD40L upon anchoring to cell surface exposed antigen via the scFv domain. RESULTS: Targeted delivery of CD40L to the carcinoma marker EpCAM on carcinoma cells induced dose-dependent paracrine maturation of DCs ~20-fold more effective than a non-targeted control scFv:CD40L fusion protein. Similarly, targeted delivery of CD40L to the B cell leukemia marker CD20 induced effective paracrine maturation of DCs. Of note, the CD20-selective delivery of CD40L also triggered loss of cell viability in certain B cell leukemic cell lines as a result of CD20-induced apoptosis. CONCLUSIONS: Targeted delivery of CD40L to cancer cells is a promising strategy that may help to trigger cancer-localized activation of CD40 and can be modified to exert additional anti-cancer activity via the targeting domain.

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.

Prognostic role of indoleamine 2,3-dioxygenase in endometrial carcinoma.

OBJECTIVE: Indoleamine-2,3-dioxygenase (IDO) suppresses the function of T-lymphocytes and is an important immune escape mechanism for cancer. Therefore, it is to be expected that IDO influences prognosis of cancer patients. This study aimed to investigate the prognostic role of IDO expression in a large cohort of endometrial carcinoma (EC) patients. METHODS: A tissue microarray containing primary EC tissue of 355 patients treated in a single institution was used to evaluate IDO expression. Expression of IDO was associated with clinicopathological characteristics, survival and previously determined numbers of CD8(+) and Foxp3(+) T-lymphocytes. RESULTS: IDO(high) expression was associated with lower numbers of intratumoral CD8(+) T-lymphocytes (p=0.031). Next to well-known prognostic parameters, IDO(high) expression was independently associated with poor disease specific survival in the general cohort of EC patients (HR 2.62, 95% C.I. 1.48-4.66, p=0.001) and among patients with early stage EC (HR 3.06, 95% C.I. 1.10-8.54, p=0.032). CONCLUSION: Our results show that IDO expression is associated with poor survival. This provides evidence that further research into the use of IDO blocking agents in cancer treatment is valid where it might be a promising new therapeutic strategy.

Infiltrating CTLs are bothered by HLA-E on tumors.

We recently described the upregulation of HLA-E in ovarian and cervical cancers. Instead of interacting with natural killer cells, HLA-E appeared to inhibit intratumoral cytotoxic T lymphocytes (CTL) via the receptor CD94/NKG2A. Strikingly, the survival benefit of intraepithelial infiltrating CTL was lost in those cancers with high HLA-E expression.

Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial.

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53-SLP) vaccine, twenty patients with recurrent elevation of CA-125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53-SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no > or = grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN-gamma producing p53-specific T-cell responses were induced in all patients who received all 4 immunizations as measured by IFN-gamma ELISPOT. An IFN-gamma secretion assay showed that vaccine-induced p53-specific T-cells were CD4(+), produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53-specific response. P53-specific T-cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53-specific T-cells. As best clinical response, stable disease evaluated by CA-125 levels and CT-scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine-induced immunity. This study shows that the p53-SLP vaccine is safe, well tolerated and induces p53-specific T-cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper-1 polarization and clinical efficacy.

Down-regulation of proteasomal subunit MB1 is an independent predictor of improved survival in ovarian cancer.

OBJECTIVE: To investigate the expression and to determine the prognostic impact of components of the antigen processing and presentation pathway (APPP) in ovarian cancer. METHODS: Expression of MB1, LMP7, TAP1, TAP2, ERp57, ERAP1, beta(2)-microglobulin and the alpha-chains, HLA-B/C and HLA-A, of the MHC class I molecules was evaluated on tissue microarrays containing primary tumor samples from 232 FIGO stages I-IV ovarian cancer patients. Expression levels were correlated to clinicopathological data and disease specific (DSS) survival. RESULTS: Patients with expression of all components of the MHC class I complex, i.e. HLA-A(+)-beta(2)-m(+) and HLA-B/C(+)-beta(2)-m(+) patients, more often had expression of LMP7, a component of the immunoproteasome than patients with other phenotypes (p<0.001). These patients were also more prone to loss of MB1, part of the constitutive multicatalytic proteasome (p<0.05). Nuclear MB1 expression was an independent predictor of worse DSS (HR 1.94, 95% CI 1.16-3.26, p=0.012). The HLA-B/C(+)-beta(2)-m(+) phenotype was an independent predictor of a better prognosis (HR 0.63, 95% CI 0.40-0.99, p=0.047). Median DSS was longer for patients with normal nuclear expression of LMP7 (57.4 vs. 31.0 months, p=0.029). CONCLUSIONS: The prognostic influence of the proteasomal subunit MB1 and the MHC class I complex in ovarian cancer provides a rationale for targeting these specific APPP components in ovarian cancer.

Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer.

PURPOSE: Ovarian cancer patients with intra-tumoral CD3(+) T-lymphocytes in primary tumor tissue have a better prognosis. This study aims to analyze the presence and relative influence of three important T-lymphocyte subsets, tumor-infiltrating CD8(+) cytotoxic T-lymphocytes (CTL), CD45R0(+) memory T-lymphocytes, and FoxP3(+) regulatory T-lymphocytes (Treg), in primary tumor tissue and omental metastases of patients with ovarian cancer. EXPERIMENTAL DESIGN: The number of CD8(+), CD45R0(+), and FoxP3(+) T-lymphocytes was determined by immunohistochemistry on a tissue micro array containing ovarian tumor tissue and/or omental metastases obtained at primary debulking surgery from 306 FIGO stage I-IV ovarian cancer patients. Immunohistochemistry data were correlated to clinicopathological parameters and survival data. RESULTS: High number of CD8(+) CTL and a high CD8(+)/FoxP3(+) ratio in ovarian-derived tumor tissue were associated with increased disease-specific survival and proved to be independent prognostic factors in multivariate analyses. In advanced stage patients, the presence of CD8(+) CTL, CD45R0(+) memory T-lymphocytes, FoxP3(+) Treg or a high CD8(+)/FoxP3(+) ratio in ovarian-derived tumor tissue was associated with an increased disease specific survival in univariate analysis, as was the presence of CD45R0(+) memory T-lymphocytes and FoxP3(+) Treg in omental metastases. Furthermore, in advanced stage patients CD8(+) cytotoxic and FoxP3(+) regulatory T-lymphocytes infiltrating ovarian-derived tumor tissue were independent predictors of increased prognosis. CONCLUSIONS: T-lymphocytes infiltrating primary and metastatic ovarian cancer sites are associated with improved prognosis. These associations are especially distinct in advanced stage patients, underlining the potential for immunotherapy as a broadly applicable therapeutic strategy.