Beyond checkpoint inhibition - Immunotherapeutical strategies in combination with radiation.

The revival of cancer immunotherapy has taken place with the clinical success of immune checkpoint inhibition. However, the spectrum of immunotherapeutic approaches is much broader encompassing T cell engaging strategies, tumour-specific vaccination, antibodies or immunocytokines. This review focuses on the immunological effects of irradiation and the evidence available on combination strategies with immunotherapy. The available data suggest great potential of combined treatments, yet also poses questions about dose, fractionation, timing and most promising multimodal strategies.

Selective, efficient modulation of activated CD4+ αßT cells by the novel humanized antibody GZ-αßTCR targeting human αßTCR.

Allograft rejection and immunosuppression are two major issues in transplantation medicine. The specific targeting of alloreactive T cells, the initiators and promoters of allograft rejection, would be a promising strategy to reduce unwanted T-cell responses and side effects of lifelong immunosuppression. The novel humanized monoclonal antibody GZ-αßTCR, specific for the human αßT-cell receptor, was tested in vitro and in vivo for its specificity and efficacy to modulate the αßT-cell compartment. GZ-αßTCR moderately induced apoptosis in resting αßT cells in vitro, an effect considerably amplified in activated T cells. A single dose of GZ-αßTCR significantly reduced human CD45(+)CD3(+) T cells in vivo, with a preferential modulation of CD4(+) αßT cells. Importantly, naive T cells, the T-cell subset from which alloreactivity emanates, were significantly reduced. Simultaneously, a significant, compensatory increase of γδ T cells was observed in vitro and in vivo in both humanized mouse models examined. GZ-αßTCR did not induce cytokines and was well tolerated. Thus, specificity and high efficacy make GZ-αßTCR a powerful tool to selectively eliminate putatively detrimental T-cell subsets, a major goal in transplantation medicine. At the same time, GZ-αßTCR spares γδ and natural killer cells, thus leaving the recipient's immune system competent for cell-mediated immunoregulation and cell-mediated immunity.

Generation, selection and preclinical characterization of an Fc-optimized FLT3 antibody for the treatment of myeloid leukemia.

The therapeutic efficacy of humanized or chimeric second-generation antitumor antibodies is clearly established, but often limited. In recent years, defined modifications of the glycosylation pattern or the amino-acid sequence of the human immunoglobulin G1 Fc part have resulted in the development of third-generation antibodies with improved capability to recruit Fc receptor-bearing effector cells. The first antibodies of this kind, currently evaluated in early clinical trials, are directed against lymphoma-associated antigens. Fc-engineered antibodies targeting myeloid leukemia are not yet available. We here report on the generation and preclinical characterization of an Fc-optimized antibody directed to the FMS-related tyrosine kinase 3 (FLT3), an antigen expressed on the leukemic blasts of all investigated patients with acute myeloid leukemia (AML). This antibody, termed 4G8SDIEM, mediated markedly enhanced cellular cytotoxicity against FLT3-expressing cell lines as well as blasts of AML patients. FLT3 expression levels on AML cells varied between 300 and 4600 molecules/cell and, in most cases, were substantially higher than those detected on normal hematopoietic precursor cells and dendritic cells (approximately 300 molecules/cell). Antibody-mediated cytotoxicity against these normal cells was not detectable. 4G8SDIEM has been produced in pharmaceutical quality in a university-owned production unit and is currently used for the treatment of leukemia patients.

Central FoxO3a and FoxO6 expression is down-regulated in obesity induced diabetes but not in aging.

BACKGROUND: Recent data suggest that insulin-like growth factor (IGF)-1 resistance in neurons prolongs longevity. In C. elegans this effect is mediated via DAF-16 the ortholog of the mammalian FoxO transcription factors. 3 different FoxO transcription factors (FoxOs) are expressed in rodent CNS: FoxO1, FoxO3a and FoxO6. METHODS: To define whether the different FoxOs are region-, sex- and age-specifically expressed, we analyzed FoxO mRNA levels in different brain regions from 6, 16, 60 and 100 weeks old mice using realtime-PCR. In addition, we fed mice a high fat diet (HFD) to experimentally induce obesity and diabetes and analyzed FoxO mRNA in the different brain regions. RESULTS: Interestingly, FoxO1 was predominantly expressed in the hippocampus whereas FoxO3a was quantitatively the most abundant FoxO in the neocortex. During aging, FoxO1 expression peaked in all brain regions at 16 weeks and FoxO6 showed its highest expression at 60 weeks in the parietal and occipital cortex. In 6 weeks old mice FoxO6 expression was higher in male compared to female mice in the hippocampus and all cortical regions. Surprisingly, in HFD animals FoxO3a was significantly less expressed in the cerebellum and all cortical regions compared to control animals. Even more dramatic, FoxO6 expression dropped about 80% in all brain regions in response to HFD. CONCLUSION: Thus, FoxOs in the CNS showed a highly distinct expression, which in addition was age- and sex-dependent. In contrast to FoxO1, FoxO3a and FoxO6 were specifically diminished in the CNS of HFD animals possibly contributing to the reduced lifespan observed in these animals.

Neuron-specific deletion of a single copy of the insulin-like growth factor-1 receptor gene reduces fat accumulation during aging.

Insulin, insulin-like growth factor-1 (IGF-1), and leptin signaling have been proposed to play an important role in regulating energy homeostasis. In order to specifically address the role of neuronal IGF-1 receptor (IGF-1R) signaling for energy expenditure and metabolism we used conditional mutagenesis. Deletion of one copy of the IGF-1R specifically in post-mitotic neurons (nIGF-1R(+/-) ) does not result in growth retardation or skeletal abnormalities. Interestingly, male nIGF-1R(+/-) mice accumulate less fat mass during aging accompanied with decreased leptin levels compared to wild-type littermates. Furthermore, male nIGF-1R(+/-) mice present with increased locomotor activity and energy expenditure. In contrast, female nIGF-1R(+/-) mice remained nearly unaffected. Circadian pattern of locomotor activity and energy expenditure as well as food and water intake did not change. Consistent with increased locomotor activity, the respiratory quotient was shifted to increased fat oxidation in nIGF-1R(+/-) mice. Surprisingly, serum IGF-1 and IGF-1 binding protein 3 (IGF-BP3) concentrations were decreased in nIGF-1R(+/-) mice despite the presence of normal pituitaries suggesting a functional feedback mechanism via neuronal IGF-1Rs, which regulate serum IGF-1 levels. Thus, we show that neuron-specific IGF-1R deletion in male mice decreases body fat accumulation and increases energy expenditure during aging.

Dipeptidyl-peptidase 4 and attractin expression is increased in circulating blood monocytes of obese human subjects.

Dipeptidyl-peptidase (DPP)-4, which catalizes the degradation of the insulinotropic incretin glucagon-like-peptide (GLP)-1, and the DPP-4 like enzyme attractin are involved in activation of T-lymphocytes and monocytes. Recently, it has been demonstrated, that the risk for certain infections is increased in type 2 diabetic patients under DPP-4 inhibitor treatment. The aim of the present study was to examine the expression of DPP-4 and attractin in circulating blood monocytes of obese and type 2 diabetic subjects. Monocytes were isolated by CD14-antibody based magnetic cell sorting from blood samples of 17 lean controls, 20 obese, non-diabetic subjects and 19 obese patients with type 2 diabetes. FACS analysis was performed to test purity of the cell preparations. Expression was measured by multiplex RT-PCR on RNA-level. DPP-4 and attractin were detectable in human circulating monocytes with attractin being expressed at higher levels compared to DPP-4. Both enzymes were significantly higher expressed in circulating blood monocytes of obese subjects compared to lean controls. In contrast, type 2 diabetes did not significantly affect expression levels. Finally, neither DPP-4 nor attractin expression was altered by sitagliptin or insulin treatment. In conclusion, our data demonstrate, that expressions of DPP-4 and attractin in circulating blood monocytes of human subjects are influenced by metabolic abnormalities with obesity being an important factor.

The role of IGF-1 receptor and insulin receptor signaling for the pathogenesis of Alzheimer's disease: from model organisms to human disease.

In different clinical studies, an association of type 2 diabetes and Alzheimer's disease (AD) has been described. However, the underlying mechanisms are still unclear. One explanation could be that vascular complications of diabetes result in neurodegeneration. Alternatively, the mechanism might be directly related to insulin and insulin-like growth factor(IGF)-1 signaling, leading to the proposal that AD is a "brain-type diabetes". Furthermore, postmortem analyses of brains from patients with AD revealed a markedly downregulated expression of insulin receptor (IR), IGF-1 receptor (IGF-1R), insulin receptor substrate (IRS)-1 and IRS-2, and these changes progress with severity of neurodegeneration. These findings raise the question, whether this phenomenon is cause or consequence of neurodegeneration. Recently, Cohen and coworkers have show that knocking down DAF-2 in C. elegans, the homolog of the mammalian IR/IGF-1R, reduces beta-amyloid(Abeta)(1-42) toxicity. Cell based experiments suggest a specific role for the IGF 1/IRS-2 signaling pathway in regulating alpha-/beta-secretase activity. Moreover circulating IGF-1 might influence Abeta clearance from the brain by promoting Abeta transport over the blood brain barrier. Interestingly, brain specific deletion of IRS-2 increases life span, suggesting that long term neuronal IGF-1R signaling might be harmful. Taken together, the data from humans and different model organisms indicate a role of IR/IGF-1R signaling in Abeta metabolism, and clearance as well as longevity. Since more studies are needed to elucidate the impact of insulin and/or IGF-1 treatment in AD, the time to propose these hormones as a potential treatment option for AD has not come yet.

Cloning of a human antibody directed against human neuroblastoma cells and specific for human translation elongation factor 1alpha.

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.

A prospective comparison of immune reconstitution in pediatric recipients of positively selected CD34+ peripheral blood stem cells from unrelated donors vs recipients of unmanipulated bone marrow from related donors.

Positively selected CD34(+) hematopoietic stem cells from unrelated donors (UD-HSCT) have been successfully transplanted, but little is known about immune reconstitution in this setting. Here we report a prospective comparison of immune reconstitution in recipients of UD-HSCT and of unmanipulated bone marrow from matched sibling donors (MSD-BMT). T-cell reconstitution occurred more than 100 days later in the UD-HSCT than in the MSD-BMT group. The first T cells after UD-HSCT were almost exclusively CD45RO(+) HLA-DR(+), whereas early-emerging T cells after MSD-BMT more frequently expressed CD62L, CD28, and CD25. In both groups, numbers of CD45RA(+) naive T cells increased after 180 days. After UD-HSCT, the T-cell-receptor (TCR)-repertoire was severely skewed and showed significantly reduced diversity during the first year, but only minor abnormalities were seen after MSD-BMT. TCR-diversity increased simultaneously with the number of naive T cells. In both groups, we observed transient expansions of gammadelta T cells. B cells were reconstituted more rapidly in UD-HSCT than in MSD-BMT recipients, whereas the rapidity of NK-cell reconstitution was similar in the two groups. In summary, T-cell reconstitution was slower after UD-HSCT than after MSD-BMT because of the delayed recovery of early memory-type T cells with reduced TCR-diversity, whereas naive T-, NK-, and B cells were reconstituted similarly in the two groups.

Human gammadelta T lymphocytes exert natural and IL-2-induced cytotoxicity to neuroblastoma cells.

Human gammadelta T lymphocytes play an important role in nonadaptive reactions to infection and early tumor defense. This is the first report that freshly isolated, native gammadelta T cells of some healthy donors can kill human neuroblastoma cells to varying degrees. Their killing ability was increased and maintained during expansion and cultivation with interleukin-2 (IL-2; 400 IU/mL) for as long as 30 days (100% specific lysis at an effector-to-target cell (E:T) ratio of 20:1). gammadelta T lymphocytes without this spontaneous killing ability gained a specific cytolytic activity of 81% +/- 10.4% SD after stimulation with IL-2 for 24 hours. gammadelta cells were isolated from peripheral blood by positive enrichment (using a magnetic cell sorting system; purity, 95.2% +/- 3.2% SD, n = 21). High natural cytotoxic activity against human neuroblastoma cell lines (>50% specific lysis at an E:T ratio of 20:1) was exhibited by one of 11 donors, whereas two of 11 showed medium cytotoxicity (30% to 50% specific lysis). Eight of 11 donors showed very slight or no lytic activity against human neuroblastoma cells (<30% specific lysis). gammadelta T cells were also cytotoxic against Daudi (32.7% specific lysis at an E:T ratio of 20:1), Raji (10.3%), Colo 205 (23.1%), A 204 (54%), K 562 (100%), and SK-N-MC (100%) cells. Isolated gammadelta T cells were grown in Iscove modified Dulbecco medium with IL-2 (400 IU/mL). Increased cell proliferation (38.5% to 182%) was induced with phytohemagglutinin, IL-15, Clodronat, OKT3, or various combinations of these. Results of cold target inhibition assays suggest a natural killer-like activity of the gammadelta T-cell killing mechanism. Peptidase or papain render neuroblastoma cells unsusceptible to gammadelta T-cell killing, suggesting the involvement of antigen peptide(s) in the process of neuroblastoma cell killing. Treatment with acid phosphatase reduced specific lysis by 66.5% +/- 34.1% SD, which suggests a binding to phosphorylated neuroblastoma cell-surface structures in the killing mechanism of gammadelta T cells. Heat shock did not affect the extent of neuroblastoma killing by gammadelta cells. Recognition of neuroblastoma cells by gammadelta cytotoxic T lymphocytes is negatively regulated by major histocompatibility complex I receptors. Evidence for natural and inducible cell cytotoxicity of gammadelta T cells against human neuroblastoma cells, easy propagation, purification, and missing alloreactivity in mixed lymphocytes cultures indicates a role for this subpopulation of T lymphocytes in adoptive immunotherapy.

Reduction of N-myc expression by antisense RNA is amplified by interferon: possible involvement of the 2-5A system.

Expression of myc-box genes can be reduced by Interferon (c-myc in Daudi cells) or Retinoic acid (N-myc in neuroblastoma cells). Interferon did not reduce N-myc expression in neuroblastoma cells. However, after transfection of the human neuroblastoma cell line LS with a vector, providing the Cadmium inducible expression of an antisense N-myc transcript, drastic reduction of N-myc RNA was achieved in these cells by incubation with Cadmium and Interferon. Treatment with Cadmium alone resulted in a comparably small reduction of N-myc transcripts in these cells. Interferon alone did not appreciably affect N-myc expression. Reduction of N-myc was accompanied with reduced cell proliferation and morphological differentiation. It is assumed that most of the inhibitory effects observed are mediated by the Interferon inducible 2-5A system.