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Chimeric antigen receptor (CAR) T cell therapy has been a great success in CD19+ hematological diseases. Natural killer (NK) CAR cells offer an alternative to CAR T cells with an intrinsic potential for universal off-the-shelf cell therapeutics. The choice of cell type and the choice of CAR are both relevant for the feasibility, effectivity, engraftment, persistence, side effects, and safety of the cell therapy. Until recently CAR NK cells have proven difficult to develop into therapeutic products. Here, we give an overview of the source of CAR NK cells, gene transfer methods, and the manufacture of CAR NK cells for clinical application. We discuss improvements, as well as future options and problems that need to be addressed.

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.

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.

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.

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.

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.

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.

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.

As polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic, we studied their interaction with human dendritic cells (hDCs). Exposure of hDCs to VLPs originating from murine (MPyV) or hamster polyomavirus (HaPyV) induced hDC maturation. In contrast, exposure of hDCs to VLPs derived from human polyomaviruses (BK and JC) and simian virus 40 (SV40) only marginally induced DC maturation. The hDCs stimulated by HaPyV- or MPyV-derived VLPs readily produced interleukin-12 and stimulated CD8-positive T-cell responses in vitro. The highest frequencies of activated T cells were again observed after pulsing with HaPyV- and MPyV-derived VLPs. Monocyte-derived hDCs both bound and internalized the various tested polyomavirus VP1-derived VLPs with different levels of efficiency, partially explaining their individual maturation potentials. In conclusion, our data suggest a high variability in uptake of polyomavirus-derived VLPs and potency to induce hDC maturation.

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.

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.

Targeting of immunological effector cells to tumor cells could be an efficient strategy of adoptive immunotherapy. The success of this strategy depends on the specificity of the effector cells and their availability in sufficient numbers. The aim of this study was to target the human natural killer cell line YT specifically to tumor cells. The cell line was modified by transfection with the cDNA of the human asialoglycoprotein receptor (ASGPR). This C-type lectin recognizes carbohydrates containing terminal galactosyl (Gal) residues, including the beta1-Gal bearing Thomsen-Friedenreich (TF) antigen, which is found on tumor cells. Binding assays revealed that the ASGPR-gene-transfected YT cell line binds significantly higher to tested target tumor cell lines than the mock-transfected control cells. Cytolytic activity against the tumor cell lines Raji, Jurkat and the TF-positive KG1 subline was increased. Genetic modification of YT cells could provide a useful tool for tumor targeting in immunotherapy.

Background EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses. Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro. These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV. In addition, mini-EBVs can be modified in E. coli to alter their genetic composition or adopt new genes. Methods We constructed a mini-EBV plasmid carrying an expression cassette for the human tumor antigen mucin encoded by the gene MUC-1. Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro. These B-cells were analyzed by FACS analyses for the expression of mucin and co-stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors. Results Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV. These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation. Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established. Conclusions Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines. Copyright © 1999 John Wiley & Sons, Ltd.

The human mucin, MUC-1, is a transmembrane glycoprotein that is produced by both normal an malignant epithelium. The MUC-1 produced by malignant epithelium is underglycosylated, which leads to the expression by tumors of novel T and B cell epitopes on the mucin polypeptide core. Similar underglycosylation occurs in the lactating breast. In this report, we describe a long-term survivor of breast cancer whose tumor strongly expressed the T- and B-cell-stimulatory epitopes. Five years after presenting with the tumor, the patient had her first pregnancy, at which time she developed fulminant lymphocytic mastitis. We demonstrate that the lactating breast produced mucin expressing the same “tumor-specific” epitopes as the original cancer. The patient had circulating anti-mucin antibodies of both the IgM and IgG isotypes (these are not found in normal controls), and mucin-specific cytotoxic T lymphocytes in the peripheral blood. Limiting – dilution analysis for mucin – specific cytotoxic T lymphocytes in three different experiments yielded frequencies of 1 in 3086, 1 in 673, and 1 in 583, compared to approximately 1 in 106 in normal controls. The patient remains clinically free of carcinoma after 5 additional years of follow-up. We propose that the original tumor primed the patient’s immune response against the mucin epitopes, and that the re-expression of these epitopes on the lactating breast evoked a secondary immune response. It is tempting to speculate that the vigor of her anti-mucin immunity may have helped protect this patient against recurrent tumor.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

The knowledge of direct effects of β-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.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.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.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-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.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.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).Beta-(1-3),(1-6)-D-glucan could be useful in the treatment of lymphoma after low-dose chemotherapy with CY.

Therapeutic options for the treatment of malignant ascites are limited and could be broadened by immune-stimulatory drugs.Soluble β-(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.The early, but not the later, administration of β-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 β-glucan at either time-point showed no effect on the ascites volume and mean survival time.β-(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.

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.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.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.The human NK-like cell line YT could be useful in immunotherapy of patients with CD86(+) multiple myeloma.

The cysteinyl leukotrienes LTC4, LTD4 and LTE4 are potent lipid mediators eliminated from the blood circulation mainly due to uptake by the liver and the kidneys. In man hepatobiliary elimination of cysteinyl leukotrienes predominates over renal excretion. In the present study, the urine from patients with extrahepatic cholestasis (n = 25) and age- and sex-matched healthy control subjects (n = 25) was analyzed for endogenous LTE4, the predominant metabolite of LTC4 excreted into urine. LTE4 was separated by reversed-phase high-performance liquid chromatography and subsequently quantified by enzyme immunoassay. Healthy subjects excreted a median concentration of 14 nmol LTE4/mol creatinine (range 5-24 nmol/mol creatinine). Its median concentration increased significantly to more than 5-fold higher levels to 74 nmol LTE4/mol creatinine (range 52-93 nmol/mol creatinine) in patients with extrahepatic cholestasis (P < 0.01). These results indicate that extrahepatic cholestasis leads to a compensatory diversion of cysteinyl leukotriene elimination to the kidney with subsequent increased excretion of LTE4 into urine.

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.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 β-glucan as adjuvant was tested in colon-26 adenocarcinoma-bearing Balb/c-mice.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.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.

Background L19-IL2, a tumour-targeting immunocytokine composed of the recombinant human antibody fragment L19 (specific to the alternatively-spliced EDB domain of fibronectin, a well characterised marker of tumour neo-vasculature) and of human IL2, has demonstrated strong therapeutic activity in animal cancer models. This phase I/II trial was performed to evaluate safety, tolerability, recommended phase II dose (RD) and early signs of activity of L19-IL2. Patients and methods Five cohorts of patients with progressive solid tumours (n = 21) received an intravenous infusion of L19-IL2 (from 5 to 30 Mio IU IL2 equivalent dose) on days 1, 3 and 5 every 3 weeks. This treatment cycle was repeated up to six times. In the following expansion phase, patients with metastatic renal cell carcinoma (RCC) (n = 12) were treated at the RD of L19-IL2. Clinical data and laboratory findings were analysed for safety, tolerability and activity. Results Preclinical studies in rats and monkeys did not raise any safety concerns. The RD was defined to be 22.5 Mio IU IL2 equivalent. Pharmacokinetics of L19-IL2 was dose proportional over the tested range, with a terminal half-life of 2–3 h. Toxicities were manageable and reversible with no treatment-related deaths. We observed stable disease in 17/33 patients (51%) and 15/18 with mRCC (83%) after two cycles. Median progression-free survival of RCC patients in the expansion phase of the study was 8 months (1.5–30.5). Conclusions L19-IL2 can be safely and repeatedly administered at the RD of 22.5 Mio IU IL2 equivalent in advanced solid tumours. Preliminary evaluation suggests clinical activity of L19-IL2 in patients with mRCC.

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.

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.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.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 (p0.05).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.

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-γ 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.

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.

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.

Dendritic cells (DC) derived from peripheral blood monocytes are currently being investigated in clinical trials for their role in stimulating the immune system. We performed a phase I/II clinical trial using human autologous DC transfected with cDNA of the human tumor antigen mucin (MUC1) as a vaccine in 10 patients with advanced breast, pancreatic or papillary cancer. After liposomal transfection, flow cytometry testing showed that 2% to 53% of the DC expressed mucin epitopes. Patients were immunized two or three times with 1 million transfected DC injected subcutaneously (s.c.). A vaccine-specific delayed-type hypersensitivity (DTH) reaction was observed in 3 out of 10 patients. After vaccination, 4 patients showed a 2- to 10-fold increase in the frequency of mucin-specific interferon-gamma (IFN-gamma)-secreting CD8+ T cells. We demonstrated the feasibility and safety of a vaccine consisting of autologous gene-transfected DC, and that immunologic responses could be induced even in patients with pretreated and advanced disease.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA-DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials.

Naked DNA is an attractive tool for vaccination studies. We have studied naked DNA vaccination against the human tumor antigen mucin, encoded by the gene MUC1. C57/BL6 mice were immunized twice, on day 1 and day 10, with plasmid pCI-MUC1, intramuscularly. Five days after the last immunization tumor challenge experiments were performed using the tumor cell line MC38, expressing human MUC1. In 85% of mice immunized with the mucin plasmid tumor growth inhibition was observed, whereas control mice developed tumors. Re-tumor challenge after three months revealed no tumor growth in mice immunized with the mucin plasmid. These encouraging results, showing long-term protection against tumor growth, indicate the potential usefulness of naked DNA vaccination for clinical immunotherapy.

Tumor antigen-specific T cell clones represent a useful tool in tumor immunology; however, their long-term culture is limited. To generate an immortalized cytotoxic T cell clone against the human tumor antigen mucin, we exposed a previously generated T cell culture to Herpesvirus saimiri. We obtained an immortalized human CD4+ T cell clone, termed SITAM. Clonality of these cells was shown by analysis of the α/β-T cell receptor (TCR) repertoire. Cytolytic activity was demonstrated against several mucin-expressing tumor cell lines and could not be detected against non-mucin-expressing cells. SITAM cells maintained their features stably for 2 years. Furthermore, growth of the tumor cell line Capan-2 in NOD/SCID mice was inhibited when SITAM cells were coinjected subcutaneously with tumor cells. SITAM cells provide an unlimited source of clonal T cells for analysis of tumor recognition and may be of help in TCR-targeted immunotherapy.

Many obstacles still stand in the way to eliciting an effective immune response against cancer, even though several antigens and antigenic peptides have been identified as potential tumor targets. All of them, including the MUC-1 mucin, share the caveat of being normal cellular proteins. Unlike all the others, however, MUC-1 expressed on tumors can still be considered a truly tumor-specific antigen. Its expression on normal cells is hidden from the immune system, and its aberrant glycosylation on tumors creates new epitopes recognized by the immune system. Moreover, all other tumor targets identified so far are MHC-restricted peptides that can only be recognized by patients who carry a specific HLA type, or on tumors which continue to express particular HLA alleles. MUC-1 is powerfully different. Recognized as a native molecule independent of MHC, it is a universal immunogen and a universal target, and if made effectively immunogenic, it would be expected to elicit immune responses in all patients, and against numerous MUC-1 expressing human tumors. It may, in fact, be the extraordinary solution to an extraordinary problem of cancer immunity and immunotherapy.