Role of beta-(1→3)(1→6)-D-glucan derived from yeast on natural killer (NK) cells and breast cancer cell lines in 2D and 3D cultures.

BACKGROUND: Beta-(1,3)(1,6)-D-glucan is a complex polysaccharide, which is found in the cell wall of various fungi, yeasts, bacteria, algae, barley, and oats and has immunomodulatory, anticancer and antiviral effects. In the present study, we investigated the effect of beta-(1,3)(1,6)-D-glucan derived from yeast on the proliferation of primary NK cells and breast cancer cell lines in 2D and 3D models, and on the cytotoxicity of primary NK cells against breast cancer cell lines in 2D and 3D models. METHODS: In this study, we investigated the effects of different concentrations of yeast-derived beta-(1→3)(1→6)-D-glucan on the proliferation and cytotoxicity of human NK cells and breast cancer cell lines in 2D and 3D models using the XTT cell proliferation assay and the CellTiter-Glo® 2.0 assay to determine the cytotoxicity of human NK cells on breast cancer cell lines in 2D and 3D models. RESULTS: We found that the co-incubation of NK cells with beta-glucan in the absence of IL2 at 48 h significantly increased the proliferation of NK cells, whereas the co-incubation of NK cells with beta-glucan in the presence of IL2 (70 U/ml) increased the proliferation of NK cells but not significantly. Moreover, beta-glucan significantly inhibited the proliferation of breast cancer cell lines in 2D model and induced a weak, non-significant growth inhibitory effect on breast cancer multicellular tumor spheroids (3D). In addition, the cytotoxicity of NK cells against breast cancer cell lines was examined in 2D and 3D models, and beta-glucan significantly increased the cytotoxicity of NK cells against MCF-7 (in 2D). CONCLUSIONS: Yeast derived beta-(1,3)(1,6)-D-glucan could contribute to the treatment of cancer by enhancing NK cell immune response as well as contributing to inhibition of breast cancer cell growth.

Next Generation CD44v6-Specific CAR-NK Cells Effective against Triple Negative Breast Cancer.

There is a medical need to develop new and effective therapies against triple-negative breast cancer (TNBC). Chimeric antigen receptor (CAR) natural killer (NK) cells are a promising alternative to CAR-T cell therapy for cancer. A search for a suitable target in TNBC identified CD44v6, an adhesion molecule expressed in lymphomas, leukemias and solid tumors that is implicated in tumorigenesis and metastases. We have developed a next-generation CAR targeting CD44v6 that incorporates IL-15 superagonist and checkpoint inhibitor molecules. We could show that CD44v6 CAR-NK cells demonstrated effective cytotoxicity against TNBC in 3D spheroid models. The IL-15 superagonist was specifically released upon recognition of CD44v6 on TNBC and contributed to the cytotoxic attack. PD1 ligands are upregulated in TNBC and contribute to the immunosuppressive tumor microenvironment (TME). Competitive inhibition of PD1 neutralized inhibition by PD1 ligands expressed on TNBC. In total, CD44v6 CAR-NK cells are resistant to TME immunosuppression and offer a new therapeutic option for the treatment of BC, including TNBC.

Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers.

Chimeric antigen receptor T (CAR-T) cells have shown promising efficacy in treatment of hematological malignancies, but its applications in solid tumors need further exploration. In this study, we investigated CAR-T therapy targeting carcino-embryonic antigen (CEA)-positive colorectal cancer (CRC) patients with metastases to evaluate its safety and efficacy. Five escalating dose levels (DLs) (1 × 105 to 1 × 108/CAR+/kg cells) of CAR-T were applied in 10 CRC patients. Our data showed that severe adverse events related to CAR-T therapy were not observed. Of the 10 patients, 7 patients who experienced progressive disease (PD) in previous treatments had stable disease after CAR-T therapy. Two patients remained with stable disease for more than 30 weeks, and two patients showed tumor shrinkage by positron emission tomography (PET)/computed tomography (CT) and MRI analysis, respectively. Decline of serum CEA level was apparent in most patients even in long-term observation. Furthermore, we observed persistence of CAR-T cells in peripheral blood of patients receiving high doses of CAR-T therapy. Importantly, we observed CAR-T cell proliferation especially in patients after a second CAR-T therapy. Taken together, we demonstrated that CEA CAR-T cell therapy was well tolerated in CEA+ CRC patients even in high doses, and some efficacy was observed in most of the treated patients.

Next-Generation CEA-CAR-NK-92 Cells against Solid Tumors: Overcoming Tumor Microenvironment Challenges in Colorectal Cancer.

Colorectal carcinoma (CRC) presents a formidable medical challenge, demanding innovative therapeutic strategies. Chimeric antigen receptor (CAR) natural killer (NK) cell therapy has emerged as a promising alternative to CAR T-cell therapy for cancer. A suitable tumor antigen target on CRC is carcinoembryonic antigen (CEA), given its widespread expression and role in tumorigenesis and metastasis. CEA is known to be prolifically shed from tumor cells in a soluble form, thus hindering CAR recognition of tumors and migration through the TME. We have developed a next-generation CAR construct exclusively targeting cell-associated CEA, incorporating a PD1-checkpoint inhibitor and a CCR4 chemokine receptor to enhance homing and infiltration of the CAR-NK-92 cell line through the TME, and which does not induce fratricidal killing of CAR-NK-92-cells. To evaluate this therapeutic approach, we harnessed intricate 3D multicellular tumor spheroid models (MCTS), which emulate key elements of the TME. Our results demonstrate the effective cytotoxicity of CEA-CAR-NK-92 cells against CRC in colorectal cell lines and MCTS models. Importantly, minimal off-target activity against non-cancerous cell lines underscores the precision of this therapy. Furthermore, the integration of the CCR4 migration receptor augments homing by recognizing target ligands, CCL17 and CCL22. Notably, our CAR design results in no significant trogocytosis-induced fratricide. In summary, the proposed CEA-targeting CAR-NK cell therapy could offer a promising solution for CRC treatment, combining precision and efficacy in a tailored approach.

Implications for Immunotherapy of Breast Cancer by Understanding the Microenvironment of a Solid Tumor.

Breast cancer is poorly immunogenic due to immunosuppressive mechanisms produced in part by the tumor microenvironment (TME). The TME is a peritumoral area containing significant quantities of (1) cancer-associated fibroblasts (CAF), (2) tumor-infiltrating lymphocytes (TIL) and (3) tumor-associated macrophages (TAM). This combination protects the tumor from effective immune responses. How these protective cell types are generated and how the changes in the developing tumor relate to these subsets is only partially understood. Immunotherapies targeting solid tumors have proven ineffective largely due to this protective TME barrier. Therefore, a better understanding of the interplay between the tumor, the tumor microenvironment and immune cells would both advance immunotherapeutic research and lead to more effective immunotherapies. This review will summarize the current understanding of the microenvironment of breast cancer giving implications for future immunotherapeutic strategies.

HCMV-Mediated Interference of Bortezomib-Induced Apoptosis in Colon Carcinoma Cell Line Caco-2.

Human cytomegalovirus (HCMV) has been implicated in the development of human malignancies, for instance in colon cancer. Proteasome inhibitors were developed for cancer therapy and have also been shown to influence HCMV infection. The aim of this study was to investigate if proteasome inhibitors have therapeutic potential for colon carcinoma and how this is influenced by HCMV infection. We show by immunofluorescence and flow cytometry that the colon carcinoma cell line Caco-2 is susceptible to HCMV infection. Growth curve analysis as well as protein expression kinetics and quantitative genome analysis further confirm these results. HCMV has an anti-apoptotic effect on Caco-2 cells by inhibiting very early events of the apoptosis cascade. Further investigations showed that HCMV stabilizes the membrane potential of the mitochondria, which is typically lost very early during apoptosis. This stabilization is resistant to proteasome inhibitor Bortezomib treatment, allowing HCMV-infected cells to survive apoptotic signals. Our findings indicate a possible role of proteasome inhibitors in colon carcinoma therapy.

Comparison of the effect of orally administered soluble beta-(1-3),(1-6)-D-glucan and of G-CSF on the recovery of murine hematopoiesis.

Beta-glucans are branched fungal polysaccharide compounds with pleiotropic activating effects on cells of the immune and the hematopoietic system. In this study, the hematopoiesis-promoting effect of an orally administered soluble beta-(1-3),(1-6)-D-glucan and of intravenously (i.v.) injected recombinant human granulocyte colony-stimulating factor (G-CSF/filgrastim) was tested in cyclophosphamide (CY)-conditioned mice. Both agents were administered for 5 consecutive days following treatment with CY. When G-CSF and the carbohydrate compound were co-administered, a small but non-significant increase of granulopoiesis compared to G-CSF alone was detected. beta-Glucan alone failed to augment granulopoiesis in the peripheral blood of CY-treated mice. However, both G-CSF and beta-glucan significantly enhanced the recovery of monocytes in the peripheral blood of leukopenic mice when orally administered as single agents. In conclusion, the present study provides further evidence of a stimulatory function of orally administered beta-glucans on monocyte production and shows a weak additive effect on granulopoiesis when co-administered with G-CSF into leukopenic mice.

Engineering NK Cells for CAR Therapy-Recent Advances in Gene Transfer Methodology.

The development of chimeric antigen receptor (CAR) T cell therapy has introduced a new and effective strategy to guide and promote the immune response against tumors in the clinic. More recently, in an attempt to enhance its utility, this method has been expanded to novel cell types. One of the more successful variants has proven to be the expression of CARs in Natural Killer (NK) cells (CAR-NK). Gene engineering NK cells to express an exogenous CAR receptor allows the innate anti-tumor ability of NK cells to be harnessed and directed against a target tumor antigen. In addition, the biology of NK cells allows the development of an allogeneic cell therapeutic product useable with most or all patient haplotypes. NK cells cause little or no graft versus host disease (GvHD) and are therefore suitable for development of an "off the shelf" therapeutic product. Initial trials have also shown that CAR-NK cells rarely cause cytokine release syndrome. However, despite their potential NK cells have proven to be difficult to engineer, with high sensitivity to apoptosis and low levels of gene expression. The creation of optimized methods to introduce genes into NK cells will promote the widespread application of CAR-NK in research laboratories and the clinics.

Cellular and humoral immunogenicity of hamster polyomavirus-derived virus-like particles harboring a mucin 1 cytotoxic T-cell epitope.

In this study, we examined hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) as a carrier for a human tumor-associated cytotoxic T lymphocyte (CTL) epitope. The VP1 tolerated the insertion of an HLA-*A2-restricted CTL epitope from human mucin 1 (MUC1) into two sites independently and simultaneously, without interfering with assembly of chimeric VLPs. Chimeric VLPs did not differ in the entry pathway or maturation potential of human dendritic cells (hDCs) compared to unmodified VLPs. Recently we demonstrated that immunization of BALB/c mice with chimeric VLPs harboring two MUC1 insertions resulted in the generation of MUC1-specific monoclonal antibodies. Here we demonstrate that the monoclonal antibodies generated react specifically with human tumor cells. Co-cultivation of chimeric VLP-primed hDCs with autologous peripheral blood leukocytes resulted in the activation of MUC1 epitope-specific CD8(+) T cells. This was evidenced by IFN-gamma secretion of an expanded MUC1-specific CD8(+) T-cell pool. The induction of epitope-specific T cells in a human in vitro model and of murine MUC1-reactive antibodies in vivo indicate the potential of chimeric HaPyV VP1-derived VLPs as a delivery vehicle for immunotherapeutic targets.

Impact of antigen-unloaded immature dendritic cells on antileukemic T-cell cytotoxicity.

BACKGROUND: Immature dendritic cells (iDC) loaded with antigens are able to induce tolerance in antigen-specific T-cells. The potential of antigen-unloaded iDC to regulate the antileukemic cytotoxicity of autologous T-cells was determined. MATERIALS AND METHODS: iDC generated with 50 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and very immature DC (viDC) generated with 10 U/ml GM-CSF from the bone marrow of Balb/c mice were used for T-cell co-culture. RESULTS: The measurement of cellular cytotoxicity against the syngeneic murine B-cell leukemia line A20 revealed that T-cells without co-culture or after co-culture with iDC exerted a similar cytotoxicity, whereas T-cells co-incubated with viDC showed a significantly diminished lysis of A20 cells (p<0.05). CONCLUSION: Antigen-unloaded iDC in contrast to antigen-loaded iDC may not affect antileukemic T-cell cytotoxicity, whereas antigen-unloaded DC cultures generated with a low dose of GM-CSF are able to impair the T-cell-mediated cytolysis of leukemic cells.

Quantification of the CD8+ T cell response against a mucin epitope in patients with breast cancer.

INTRODUCTION: Mucin 1, encoded by the MUC1 gene, is a tumor-associated antigen expressed on the surface of breast cancer cells. It would be of interest to see whether there is a naturally existing T cell immune response against mucin epitopes in cancer patients. MATERIALS AND METHODS: Using tetramer and interferon-gamma assays, the immune response to one MUC1 peptide epitope in the peripheral blood of breast cancer patients was quantified. The data were compared with the clinical course of the patients. RESULTS: CD8(+) T cells capable of recognizing the HLA-A*0201-restricted STAPPVHNV epitope were detected in 9 of 19 patients with a frequency ranging 0.01-0.082%. No significant difference was found between the occurrence of epitope-specific CD8(+) T cells of patients with progressive disease and disease-free patients. However, all patients with stable disease showed a specific immune response, including both patients with the highest frequency. CONCLUSIONS: The results of this study provide further evidence that a natural specific cellular immune response against this mucin epitope exists in breast cancer patients.

Efficacy and toxicity of docetaxel combination chemotherapy for advanced squamous cell cancer of the head and neck.

The aim of the present study was to evaluate the clinical effectiveness and toxicity of docetaxel with 5-fluorouracil and cisplatin as combination treatment in patients with curable or metastatic/recurrent head and neck cancer by a retrospective cohort study of patients treated at a single institution between 2007 and 2012. Patients with locally advanced, metastatic and/or recurrent squamous cell carcinoma of the head and neck (SCCHN), who were treated with a combination therapy including docetaxel, were considered as eligible. Survival data, clinical side effects, quality of life (QoL) and toxicity profile were retrieved from patient charts, analyzed and scored according to the National Cancer Institute Common Toxicity Criteria, version 4, and the Response Evaluation Criteria In Solid Tumors, version 1.1. An overall response rate of 86% and a 3-year survival of 65.1% were observed. The median progression-free survival was 32 months. The cumulative incidence after 3 years was 16.9% for local recurrence and 10.4% for distant metastasis. Leukopenia (58%) and anemia (51%) were the most common hematological toxicities, followed by hepatotoxicity (53%) and nausea (27%). A total of 31% of the patients experienced a compromise in their QoL following therapy completion. In conclusion, docetaxel in combination with cisplatin and 5-fluorouracil was found to effectively prolong survival in patients with locally advanced and/or recurrent metastatic SCCHN. The overall survival, progression-free survival and response rates were in accordance with those reported by previous clinical trials. Therefore, this therapy protocol is recommended for patients with SCCHN in the curative as well as the palliative settings.

Specific targeting of CD33(+) leukemia cells by a natural killer cell line modified with a chimeric receptor.

We directed the human natural killer (NK) cell line YT by gene transfer of a humanized chimeric immunoglobulin T cell receptor to CD33, a marker on myeloid leukemias. The chimeric receptor was generated using a CD33 specific single-chain Fv (scFv) fragment based on the humanized antibody HuM195, the human IgG1 Fc domains and the human CD3 zeta signal chain. YT cells transfected by electroporation with the chimeric receptor gene specifically lysed the acute myeloid leukemia (AML) cell line KG1. This gene-modified NK cell line available in unlimited source could be an attractive tool in immunotherapy.

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo.

The gene transfer of tumor-specific chimeric immunoglobulin T-cell receptors (cIgTCRs) combining antibody-like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human natural killer (NK) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the human carcinoembryonic antigen (CEA). The cIgTCR was constructed of a CEA-specific humanized single-chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 zeta chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR-expressing cells were enriched by immunoaffinity purification. cIgTCR-expressing YT cells specifically lysed CEA(+) colon carcinoma cell lines, which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. Receptor gene-modified YT cells retained their CEA-specific cytolytic activity after gamma-irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into NOD/SCID mice. This gene-modified NK cell line available in unlimited source might be useful in clinical immunotherapy of CEA(+) cancer.

Tumor growth inhibition elicited by different vaccines and correlation with antigen specific cytotoxic T-cell frequencies determined by intracellular interferon-gamma staining.

Different vaccines based on naked DNA and the modified vaccinia virus Ankara (MVA) were compared for their efficiency to protect mice against tumors bearing the model antigen beta-galactosidase (beta-Gal) and for their potential to induce an antigen specific cellular immune response. Mice were immunized with the LacZ gene applied as naked DNA. In accordance with the observed beta-Gal-specific T-cell frequency, only 20% of mice boosted with LacZ naked DNA developed tumors whereas all mice boosted with MVA expressing LacZ developed a tumor. Mice vaccinated with mock DNA or mock virus developed tumors in 60 or 100%, respectively. MVA vaccination led to strong and long-lasting CD4- and CD8-T-cell responses against viral antigens but not against beta-Gal.

Down-regulation of the human tumor antigen mucin by gemcitabine on the pancreatic cancer cell line capan-2.

The nucleoside analogue gemcitabine displays therapeutic effects mainly against breast, ovarian and pancreatic cancer. Mucin, encoded by the gene MUC1, is a well-established tumor antigen expressed on these tumors. Knowledge of possible effects of chemotherapeutic drugs on the level of mucin epitope expression is important for the design of clinical protocols combining chemo- and immunotherapeutic approaches. In this study, we determined the influence of gemcitabine on the mucin expression of the human pancreatic carcinoma cell line Capan-2. The cells were treated with three different concentrations (0.01 microg/ml, 0.1 microg/ml and 0.25 microg/ml) of gemcitabine or were left untreated and were analyzed after 24 hours, 3 and 5 days. Flow cytometric analysis showed a dose-dependent decrease of mucin expression on the cell surface which remained over 5 days. The strongest reduction of mucin expression was detectable 24 hours after application of the drug. The down-regulation of the tumor antigen mucin by gemcitabine might weaken an immune response against mucin-expressing tumors, which are under treatment with this chemotherapeutic drug.

Pattern of the epitope-specific IgG/IgM response against human cytomegalovirus in patients with multiple myeloma.

Human cytomegalovirus (HCMV) is a member of the herpesvirus family and represents a major human pathogen causing severe disease in newborns and immunocompromised patients, e.g., organ transplant recipients and patients with AIDS. One characteristic of herpesviruses is their ability to establish lifelong latency in their hosts; thus, reactivation during immunosuppression leads to recurrent episodes of disease. In several recent reports, it has been shown that HCMV infection may occur in patients with malignancy. This study focused on HCMV infection in patients with multiple myeloma (MM). In order to determine the IgM and IgG humoral immune response, sera from MM patients and healthy donors were analyzed with an accredited immunoblot test, and the IgM response was analyzed with an accredited enzyme-linked immunosorbent assay. A response against HCMV was detected in 80% of the MM patients. While the IgG pattern varied in each patient, the most prominent IgM response was against the tegument protein pp150 and two nonstructural proteins, the processivity factor (pUL44) and the single-stranded DNA binding protein (pUL57). An IgG avidity test revealed that 4 out of 20 MM patients had a fresh infection and 2 MM patients had a recent infection. The combination of IgG avidity and the IgM pattern will be a useful tool for reliable clinical diagnostics concerning HCMV and for application of early therapy for those MM patients suffering from a high viral load.

Natural killer cell line YT exerts cytotoxicity against CD86+ myeloma cells.

BACKGROUND: Human NK cell lines providing an unlimited source of effector cells might be suitable for use in adoptive immunotherapy. This study determined the cytolytic activity of the human NK-like cell line YT against myeloma cell lines and primary myeloma cells. MATERIALS AND METHODS: Lysis of the myeloma cell lines MM1S and U266 and of primary human myeloma cells by YT was measured using a flow-cytometric cytotoxicity assay. Furthermore, it was investigated whether the cytotoxicity correlates with the expression of CD86 on myeloma cells and the effect of different doses of IL-2 on cytolysis was tested. RESULTS: YT showed killing of myeloma cell lines and primary myeloma cells. The extent of cytolysis correlated with the expression of CD86 on myeloma cells and was not augmented by preincubation of YT with high dose of IL-2. CONCLUSION: The human NK-like cell line YT could be useful in immunotherapy of patients with CD86(+) multiple myeloma.

Beta-(1-3),(1-6)-D-glucan enhances the effect of low-dose cyclophosphamide treatment on A20 lymphoma in mice.

BACKGROUND: Beta-(1-3),(1-6)-D-glucans demonstrate antitumor effects in vivo due to the activation of innate immune cells. Cyclophosphamide (CY) enhances natural or therapeutically induced antitumor immune responses by reducing the number and activity of regulatory T (Treg) cells. MATERIALS AND METHODS: We tested whether oral administration of soluble beta-glucan augmented the inhibitory effect of intraperitoneally injected low-dose CY (30 mg/kg) on subcutaneously growing A20-lymphoma in Balb/c-mice. RESULTS: Administration of CY one week after tumor inoculation significantly diminished tumor growth (p=0.009) and the absolute number of Treg cells in the peripheral blood compared with phosphate buffered saline-treated mice (p=0.036). Treatment of CY pre-conditioned lymphoma-bearing mice with daily beta-glucan (400 µg/mouse) between day 9 and day 13 after tumor injection significantly delayed onset of tumor growth, compared to mice which received only CY (p=0.01). CONCLUSION: Beta-(1-3),(1-6)-D-glucan could be useful in the treatment of lymphoma after low-dose chemotherapy with CY.

Role of soluble ß-(1-3),(1-6)-D-glucan from Saccharomyces cerevisiae in the murine P388 ascites tumor model.

BACKGROUND: Therapeutic options for the treatment of malignant ascites are limited and could be broadened by immune-stimulatory drugs. MATERIALS AND METHODS: Soluble ß-(1-3),(1-6)-D-glucan from Saccharomyces cerevisiae was administered i.p. into DBA/2-mice bearing the P388 lymphoma either freshly inoculated or as an established ascites-tumor. Its effect on survival, ascites volume and production of cytokines was examined. RESULTS: The early, but not the later, administration of ß-glucan showed a tendency to induce interleukin (IL)-12 in the ascites, whereas both treatment schedules demonstrated a clear tendency to reduce production of interferon-γ in the abdominal fluid and had no notable impact on the level of tumor necrosis factor-α. Treatment with ß-glucan at either time-point showed no effect on the ascites volume and mean survival time. CONCLUSION: ß-(1-3), (1-6)-D-Glucan shows weak and differential modulation of immune-stimulatory and pro-inflammatory cytokines in tumor ascites dependent on the stage of tumor growth without affecting survival of the mice.