Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts.

The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis plays a central role in suppression of anti-tumor immunity. Blocking the axis by targeting PD-L1 with monoclonal antibodies is an effective and already clinically approved approach to treat cancer patients. Glyco-engineering technology can be used to optimize different properties of monoclonal antibodies, for example, binding to FcγRs. We generated two glycosylation variants of the same anti-PD-L1 antibody: one bearing core fucosylated N-glycans in its Fc part (92%) and its de-fucosylated counterpart (4%). The two glycosylation variants were compared to a non-glycosylated commercially available anti-PD-L1 antibody in various assays. No differences were observed regarding binding to PD-L1 and blocking of this interaction with its counter receptors PD-1 or CD80. The de-fucosylated anti-PD-L1 antibody showed increased FcγRIIIa binding resulting in enhanced antibody dependent cellular cytotoxicity (ADCC) activity against PD-L1+ cancer cells compared to the "normal"-glycosylated variant. Both glycosylation variants showed no antibody-mediated lysis of B cells and monocytes. The non-glycosylated reference antibody showed no FcγRIIIa engagement and no ADCC activity. Using mixed leukocyte reaction it was observed that the de-fucosylated anti-PD-L1 antibody induced the strongest CD8 T cell activation determined by expression of activation markers, proliferation, and cytotoxicity against cancer cells. The systematic comparison of anti-PD-L1 antibody glycosylation variants with different Fc-mediated potencies demonstrates that our glyco-optimization approach has the potential to enhance CD8 T cell-mediated anti-tumor activity which may improve the therapeutic benefit of anti-PD-L1 antibodies.

Antibody-based delivery of tumor necrosis factor (L19-TNFα) and interleukin-2 (L19-IL2) to tumor-associated blood vessels has potent immunological and anticancer activity in the syngeneic J558L BALB/c myeloma model.

PURPOSE: To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice. METHODS: PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20 h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells. RESULTS: Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20 h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4- CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (- 50%), and an increase in PD-L1+ myeloma cells. CONCLUSION: IL2, TNFα, or IL2 plus TNFα (final 20 h) increased the proportion of CD4- CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.

Yeast-derived beta-(1-3),(1-6)-D-glucan induces up-regulation of CD86 on dectin-1-positive human B-lymphoma cell lines.

BACKGROUND: The knowledge of direct effects of ß-glucans on tumor cells is limited. This study evaluated the impact of a soluble yeast-derived beta-(1-3),(1-6)-d-glucan, containing a fraction of aggregated sugar polymers, on viability, proliferation and expression of CD86 of the human B-cell lymphoma cell lines Daudi and Raji. MATERIALS AND METHODS: Proliferation of carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained cell lines was determined by measuring depletion of the dye and cell death was quantified by staining with propidium iodide, both by flow cytometry. Surface expression of CD86 and the beta-glucan receptors dectin-1 and complement receptor 3 (CR3) was assessed by flow cytometry. RESULTS: Exposure to the carbohydrate increased the expression of CD86 on both dectin-1(+)CR3(-) cell lines, whereas proliferation and viability of the cells were not affected. CONCLUSION: Yeast-derived beta-glucan lacks cytotoxic effects towards B-lymphoma cells but up-regulation of CD86 suggests maturation of the cells via dectin-1 by the carbohydrate.

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.

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.

IL-1 receptor antagonist anakinra enhances tumour growth inhibition in mice receiving peptide vaccination and beta-(1-3),(1-6)-D-glucan.

BACKGROUND: Immunotherapy of cancer by vaccination is hampered by tumour-mediated immune suppression, to which pro-inflammatory cytokines such as interleukin-1 (IL-1) and IL-6 contribute. In mouse models, IL-1-receptor antagonist (IL-1 Ra) diminished inflammation and tumour growth when administered during or shortly after tumour inoculation. MATERIALS AND METHODS: The capacity of IL-1 Ra anakinra to reduce IL-1-induced production of IL-6 in order to improve the efficacy of a subsequent booster vaccination with survivin-derived peptides and soluble ß-glucan as adjuvant was tested in colon-26 adenocarcinoma-bearing Balb/c-mice. RESULTS: Bolus administration of anakinra into non-immunized mice with macroscopic tumour significantly lowered serum levels of IL-6 without inhibiting tumour growth. When administered to pre-immunized mice bearing a palpable tumour, IL-1 Ra enhanced growth inhibition of a subsequent booster vaccination, although serum-IL-6 was not reduced and the number of IFN-γ-producing splenic CD8(+) T-cells was not increased. CONCLUSION: Anakinra contributes to growth-inhibition of small tumours, presumably by blocking IL-1 as tumour growth-promoting factor rather than by facilitating an enhanced CTL response.

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.

Oral administration of a soluble 1-3, 1-6 beta-glucan during prophylactic survivin peptide vaccination diminishes growth of a B cell lymphoma in mice.

beta-glucans are biological response modifiers with activatory effects on macrophages, dendritic cells (DC), granulocytes and NK cells. In this study, we investigated the effect of a soluble yeast-derived beta-(1-3), (1-6)-D-glucan on prophylactic peptide vaccination against the B cell lymphoma A20 in syngeneic Balb/c mice. We found that repeated immunizations with two MHC class-I restricted peptides derived from the tumor antigen survivin combined with oral co-administration of beta-glucan could significantly diminish intradermal tumor growth, whereas peptide vaccination alone failed to control tumor growth. beta-glucan as single agent induced only a weak but non-significant growth inhibitory effect. To determine whether the tumor inhibitory effect of the combined treatment was associated with the induction of a tumor-specific immune response we quantified splenic DC and macrophages, analyzed the maturation of DC and measured the frequency of peptide-specific CD8+ and CD4+ T cells. Treated mice showed significantly increased numbers of splenic macrophages and mature DC compared to untreated tumor-bearing mice. After restimulation with both peptides in vitro elevated levels of interferon (IFN)-gamma-secreting CD8+ T cells were found in two of four tested mice following treatment and one of four mice showed a strong increase of interleukin (IL)-4-secreting CD4+ T cells. Our data reveal a beneficial effect of beta-(1-3), (1-6)-D-glucan in tumor growth inhibition by tumor-specific peptide vaccination which may rely on a function of the polymeric sugar as immunological adjuvant.

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.

Efficient gene transfer into murine embryonic stem cells by nucleofection.

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.

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.