Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

Dissecting the Immune Stimulation Promoted by CSF-470 Vaccine Plus Adjuvants in Cutaneous Melanoma Patients: Long Term Antitumor Immunity and Short Term Release of Acute Inflammatory Reactants.

As cutaneous melanoma (CM) currently remains with a bleak prognosis, thorough investigation of new treatment options are of utmost relevance. In the phase II/III randomized clinical trial (CASVAC-0401), the repeated immunization of stages IIB-III CM patients with the irradiated, allogeneic cellular CSF-470 vaccine plus the adjuvants bacillus Calmette-Guerin (BCG) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) demonstrated a significant benefit over IFN-alpha2B treatment in distant metastasis-free survival. Here we present on the short and long term immune monitoring results after completing the 2-year protocol; a continuation of the previous report by Mordoh et al. (1). We demonstrate that the repeated CSF-470 vaccinations stimulated a long term cellular and humoral immunity response directed against the vaccine antigens. In the case of 2 patients, we are able to show that a similar immune response was generated against autologous antigens. Evaluation of inhibitory receptor co-expression on patient's T cells indicates that the vaccination protocol did not stimulate T cell exhaustion. In order to better understand the basis for the efficacious vaccine responses observed, we investigated the short term immune events following vaccine injection. A significant increase in C-reactive protein (CRP) and IL-6 was observed 24 h after vaccination, with in vitro studies suggesting IL-6 production occurs in the vaccine site. We demonstrate that CRP enhances the cytotoxicity of peripheral blood mononuclear cells (PBMC) against melanoma cells in an in vitro model. Additionally, CRP stimulates the release of pro and anti-inflammatory cytokines from PBMC. As our results demonstrate that successive vaccinations with CSF-470 plus adjuvants promoted an increase in both anti-tumor innate and adaptive immunity, we propose a subsequent model of action.

Early Events of the Reaction Elicited by CSF-470 Melanoma Vaccine Plus Adjuvants: An In Vitro Analysis of Immune Recruitment and Cytokine Release.

In a previous work, we showed that CSF-470 vaccine plus bacillus Calmette-Guerin (BCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) as adjuvants resulted in a significant benefit in the distant metastasis-free survival when comparing vaccinated vs. IFN-α2b-treated high-risk cutaneous melanoma patients in a Phase II study. Immune monitoring demonstrated an increase in anti-tumor innate and adaptive immunities of vaccinated patients, with a striking increase in IFN-γ secreting lymphocytes specific for melanoma antigens (Ags). In an effort to dissect the first steps of the immune response elicited by CSF-470 vaccine plus adjuvants, we evaluated, in an in vitro model, leukocyte migration, cytokine production, and monocyte phagocytosis of vaccine cells. Our results demonstrate that leukocytes recruitment, mostly from the innate immune system, is an early event after CSF-470 vaccine plus BCG plus GM-CSF interaction with immune cells, possibly explained by the high expression of CCL2/MCP-1 and other chemokines by vaccine cells. Early release of TNF-α and IL-1ß pro-inflammatory cytokines and efficient tumor Ags phagocytosis by monocytes take place and would probably create a favorable context for Ag processing and presentation. Although the presence of the vaccine cells hampered cytokines production stimulated by BCG in a mechanism partially mediated by TGF-ß and IL-10, still significant levels of TNF-α and IL-1ß could be detected. Thus, BCG was required to induce local inflammation in the presence of CSF-470 vaccine cells.

Cytokine-enhanced maturation and migration to the lymph nodes of a human dying melanoma cell-loaded dendritic cell vaccine.

Dendritic cells (DCs) are professional APCs used for the development of cancer vaccines because of their ability to activate adaptive immune responses. Previously, we designed the DC/Apo-Nec vaccine using human DCs loaded with dying melanoma cells that primed Ag-specific cytotoxic T cells. Here, we evaluate the effect of a standard pro-inflammatory cytokine cocktail (CC) and adjuvants on DC/Apo-Nec maturation and migration. CC addition to the vaccine coculture allowed efficient Ag uptake while attaining strong vaccine maturation with an immunostimulatory profile. The use of CC not only increased CCR7 expression and the vaccine chemokine responsiveness but also upregulated matrix metalloproteinase-9 secretion, which regulated its invasive migration in vitro. Neither IL-6 nor prostaglandin E2 had a negative effect on vaccine preparation. In fact, all CC components were necessary for complete vaccine maturation. Subcutaneously injected DC/Apo-Nec vaccine migrated rapidly to draining LNs in nude mice, accumulating regionally after 48 h. The migrating cells of the CC-matured vaccine augmented in proportion and range of distribution, an effect that increased further with the topical administration of imiquimod cream. The migrating proportion of human DCs was detected in draining LNs for at least 9 days after injection. The addition of CC during DC/Apo-Nec preparation enhanced vaccine performance by improving maturation and response to LN signals and by conferring a motile and invasive vaccine phenotype both in vitro and in vivo. More importantly, the vaccine could be combined with different adjuvants. Therefore, this DC-based vaccine design shows great potential value for clinical translation.

Overexpression of CD85j in TNBC patients inhibits Cetuximab-mediated NK-cell ADCC but can be restored with CD85j functional blockade.

Clinical studies suggest that triple negative breast cancer (TNBC) patients with epidermal growth factor receptor (EGFR)-expressing tumors could benefit from therapy with Cetuximab, which targets EGFR. NK cells are the primary effectors of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) and thus play a role in Ab-based therapies. We have previously described diminished levels of Cetuximab-mediated ADCC in vitro in patients with advanced breast cancer. Here, we investigated the potential causes of this NK-cell functional deficiency. We characterized NK-cell activating/inhibitory receptors in the peripheral blood of breast cancer patients and found CD85j inhibitory receptor overexpression. The capacity of NK cells to perform Cetuximab-triggered ADCC against TNBC cells correlated inversely with CD85j expression, even in the presence of the stimulatory cytokines IL-2 or IL-15. Hence, patients expressing high levels of CD85j had an impaired ability to lyse TNBC cells in the presence of Cetuximab. We also found that CD85j overexpression was associated with HLA-I and soluble HLA-G expression by tumors. A CD85j functional blockade with a CD85j antagonist Ab restored ADCC levels in breast cancer patients and reverted this negative effect. Our data suggest that strategies that overcome the hurdles of immune activation could improve Cetuximab clinical efficacy.

High lipid content of irradiated human melanoma cells does not affect cytokine-matured dendritic cell function.

Gamma irradiation is one of the methods used to sterilize melanoma cells prior to coculturing them with monocyte-derived immature dendritic cells in order to develop antitumor vaccines. However, the changes taking place in tumor cells after irradiation and their interaction with dendritic cells have been scarcely analyzed. We demonstrate here for the first time that after irradiation a fraction of tumor cells present large lipid bodies, which mainly contain triglycerides that are several-fold increased as compared to viable cells as determined by staining with Oil Red O and BODIPY 493/503 and by biochemical analysis. Phosphatidyl-choline, phosphatidyl-ethanolamine and sphingomyelin are also increased in the lipid bodies of irradiated cells. Lipid bodies do not contain the melanoma-associated antigen MART-1. After coculturing immature dendritic cells with irradiated melanoma cells, tumor cells tend to form clumps to which dendritic cells adhere. Under such conditions, dendritic cells are unable to act as stimulating cells in a mixed leukocyte reaction. However, when a maturation cocktail composed of TNF-alpha, IL-6, IL-1beta and prostaglandin E2 is added to the coculture, the tumor cells clumps disaggregate, dendritic cells remain free in suspension and their ability to efficiently stimulate allogeneic lymphocytes is restored. These results help to understand the events following melanoma cell irradiation, shed light about interactions between irradiated cells and dendritic cells, and may help to develop optimized dendritic cell vaccines for cancer therapy.

IL-2- or IL-15-activated NK cells enhance Cetuximab-mediated activity against triple-negative breast cancer in xenografts and in breast cancer patients.

Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.

MART-1- and gp100-expressing and -non-expressing melanoma cells are equally proliferative in tumors and clonogenic in vitro.

MART-1 and gp100 are prototypical melanoma antigen (Ag), but their clinical use as vaccines or as targets of cytotoxic lymphocytes achieved modest success. Possible explanations could be that as MART-1 and gp100 are melanocyte differentiation Ag, clonogenic Ag-non-expressing cells would be spared by immune effectors, or that clonogenic cells would be intrinsically resistant to cytotoxic lymphocytes. We therefore analyzed the proliferative status of MART-1/gp100-expressing and -non-expressing cells in biopsies, and the clonogenicity and sensitiveness to cytotoxic lymphocytes of the human cutaneous melanoma cell lines MEL-XY1 and MEL-XY3. Analysis of MART-1/gp100 and Ki-67 expression in 22 melanoma tumors revealed that MART-1/gp100-expressing and -non-expressing cells proliferated competitively. MART-1, gp100, tyrosinase, and CD271 expression were studied in MEL-XY1 and MEL-XY3 colonies. At 7 days, colonies displayed positive, negative, and mixed expression patterns. By 14 days, colonies of different sizes developed, showing cells with different clonogenic potential, and Ag were downregulated, suggesting Ag plasticity. Subcloning of MEL-XY1 colonies showed that Ag expression varied with time without interfering with clonogenicity. Finally, clonogenic, MART-1/gp100-expressing cells were lysed by specific CD8 lymphocytes. Thus, MART-1 and gp100 expression and plasticity would not interfere with proliferation or clonogenicity, and clonogenic cells may be lysed by cytotoxic lymphocytes.

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity.

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.

A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression.

BACKGROUND: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. METHODS: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC. RESULTS: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed. CONCLUSION: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis. TRIAL REGISTRATION: Clinicaltrials.gov (NHI) NCT00515983.

Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8(+) T lymphocytes.

BACKGROUND: In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells). METHODS: We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100. RESULTS: Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change. CONCLUSION: We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.

A phase I study of an allogeneic cell vaccine (VACCIMEL) with GM-CSF in melanoma patients.

We investigated whether recombinant human granulocyte-monocyte-colony-stimulating factor (rhGM-CSF) increased the immunogenicity of VACCIMEL, a vaccine consisting of 3 irradiated allogeneic melanoma cell lines. A phase I clinical trial was performed on 20 melanoma patients in stages IIB (n=2), III (n=10), and IV (n=8), who were disease free after surgery (n=16) or had minimal disease (n=4). Cohorts of 4 patients were vaccinated 4 times with VACCIMEL and bacillus Calmette Guerin (BCG) as adjuvant. Besides, the patients received placebo (group 1) or GM-CSF: 150 microg (group 2), 300 microg (group 3), 400 microg (group 4), and 600 microg (group 5) per vaccine. The combination of VACCIMEL and GM-CSF had low toxicity. Only in group 5, grade 2 thoracic pain (3/4 patients) and abdominal cramps (2/4 patients) were observed. Delayed-type hypersensitivity increased after vaccination and it was highest in group 4. Phytohemagglutinin stimulation of peripheral blood lymphocytes was analyzed in 9 patients: 4/9 had normal stimulation; 3/9 had low basal stimulation, which recovered after vaccination; and 2/9 were not stimulated. Antimelanoma antibodies preexisted in 9/19 patients; in 3/19 patients, antibodies anti-33 kd, 90 kd, and 100 kd antigens were induced by vaccination. IgG2 but not IgG1 antibodies were detected. Anti-BCG antibodies, mostly IgG2, reached the highest post/prevaccination ratio in group 4. Median serum interleukin-12 was lower in progressing patients (61.6 pg/mL) than in those without evident disease (89 pg/mL). Thus, its low toxicity and the induction of a predominantly cellular immune response suggest that the addition of 300 to 400 microg GM-CSF to VACCIMEL is useful in increasing the immune response.