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The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1alpha. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

In this article, we increased the sensitivity of the heat-transfer method (HTM) for the detection of breast cancer cells (ZR-75-1 cells, see figure) in phosphate buffered saline (PBS). The effect of small technological changes on the limit of detection (LoD) of the methodology was examined. To this extent, polished aluminum substrates with a mirror finish were used, replacing the unpolished chips used in previous studies. These chips were coated with a polyurethane layer and imprinted for the target cell type, creating a so-called surface imprinted-polymer (SIP). Binding of target cells to the SIP resulted in an increase of the thermal resistance at the solid-liquid interface under study. Background thermal resistance measurements were performed with polished and unpolished aluminum substrates. In addition, the effect of using silver paste as thermal coupling between the aluminum chip and the copper heat provider was analyzed. The results of these experiments reveal that optimal thermal contact is achieved when directly coupling the copper heat provider to the polished side of the aluminum substrate as evidenced by a decrease in the baseline thermal resistance. In addition, noise levels on the heat-transfer resistance (R-th) signal decreased by a factor in the optimal configuration. Dose-response curves were obtained using the optimized methodology and were compared with results obtained with the original substrates. These quantitative experiments demonstrated an improvement of the LoD by approximately thirty percent. ZR-75-1 cells applied onto a home-made rubber stamp.

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA). Methods: The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA (from cetuximab) and scFv1711-ETA (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-ta g imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. Results: Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. Conclusions: These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of Fc alpha RI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.

Immunotoxins are fusion proteins that combine a targeting component such as an antibody fragment or ligand with a cytotoxic effector component that induces apoptosis in specific cell populations displaying the corresponding antigen or receptor. Human cytolytic fusion proteins (hCFPs) are less immunogenic than conventional immunotoxins because they contain human pro-apoptotic enzymes as effectors. However, one drawback of hCFPs is that target cells can protect themselves by expressing endogenous inhibitor proteins. Inhibitor-resistant enzyme mutants that maintain their cytotoxic activity are therefore promising effector domain candidates. We recently developed potent variants of the human ribonuclease angiogenin (Ang) that were either more active than the wild-type enzyme or less susceptible to inhibition because of their lower affinity for the ribonuclease inhibitor RNH1. However, combining the mutations was unsuccessful because although the enzyme retained its higher a ctivity, its susceptibility to RNH1 reverted to wild-type levels. We therefore used molecular dynamic simulations to determine, at the atomic level, why the affinity for RNH1 reverted, and we developed strategies based on the introduction of further mutations to once again reduce the affinity of Ang for RNH1 while retaining its enhanced activity. We were able to generate a novel Ang variant with remarkable in vitro cytotoxicity against HL-60 cells and pro-inflammatory macrophages. We also demonstrated the pro-apoptotic potential of Ang-based hCFPs on cells freshly isolated from leukemia patients.

We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B.

The treatment of rhabdomyosarcoma (RMS) is challenging, and the prognosis remains especially poor for high-grade RMS with metastasis. The conventional treatment of RMS is based on multi-agent chemotherapy combined with resection and radiotherapy, which are often marked by low success rate. Alternative therapeutic options include the combination of standard treatments with immunotherapy. We generated a microtubule-associated protein (MAP)-based fully human cytolytic fusion protein (hCFP) targeting the fetal acetylcholine receptor, which is expressed on RMS cells. We were able to express and purify functional scFv35-MAP from Escherichia coli cells. Moreover, we found that scFv35-MAP is rapidly internalized by target cells after binding its receptor, and exhibits specific cytotoxicity toward FL-OH1 and RD cells in vitro. We also confirmed that scFv35-MAP induces apoptosis in FL-OH1 and RD cells. The in vivo potential of scFv35-MAP will need to be considered in further studies.

As perennials, forage crops are more closely exposed to climate change than many other crops. Whilst a global elevation of temperature may appear favourable to higher productivity, higher temperatures are generally associated with more frequent and/or intense water deficits in summer. In addition, the expectations of IPCC (Intergovernmental Panel on Climate Change) stress that climate change will include more incidents of extreme and fluctuating weather events including episodes of heavy rainfall especially in Autumn and lower temperatures in Winter. [br/]In this context, forage grass breeders and geneticists have long been developing an inter(-generic)–specific hybridization approach to attribute genomic plasticity to varieties capable of providing adaptations to contrasting climate conditions sufficient to ensure consistent and sustainable forage production at different locations.[br/]Festulolium include all natural and artificial hybrids between the two genera of perennial grasses, Festuca (fescue) and Lolium (ryegrass). As synthetics, current Festulolium varieties involve one of the two ryegrass species (L. perenne, L; multiflorum) with any one of the three interrelated fescue species (F. arundinacea, F. pratensis and F. glaucescens); they may also exist under a range of quite diverse genome constitution including allotetraploid combinations and introgression-lines.[br/]A 21-entries trial of Festulolium and controls of pure species has been underway since 2012 in eight European countries, from oceanic and continental climate longitudinally as well as from cold Mediterranean to Nordic climate latitudinally. Forage productivity is currently assessed by a 3-5 cuts a year management depending on local seasonal growth. Feeding value at reproductive growth in spring is estimated through NIRS predictions of Nitrogen, lignin and water soluble carbohydrate content in dry matter. Disease susceptibility, persistency and recovery of the plots are also scored at the crucial times of the year following strong climatic stresses.[br/]It is expected that the stress tolerance traits, which are widely distributed among parental species, and included at various degree in Festulolium, will be clarified in their respective roles on long term persistency and pasture productivity. Given the IPCC expectations for the near future and the constant advance in molecular tools and cytogenetics, outcomes of the field trials will help better define realistic ideotypes of grass varieties and will provide a guide to future plant breeding strategies.

During leukemia, hematologic precursor cells suffer from an inheritable cancerogenic event (e.g. mutation), resulting in their dysfunction, buildup of immature cells and an imbalanced hematopoietic system. For future diagnosis and treatment, specificity of probes and therapeutics will be of paramount importance. Thus, our highly specific constructs for cell surface markers of leukemic blasts and leukemic stem cells are excellent therapeutic and diagnostic candidates. To further improve the internalization and cytotoxicity, we created bivalent constructs and cytolytic fusion proteins with human effector domains for subset of constructs. We intend to enhance internalization by crosslinking of cell surface receptors upon binding. Additionally, we aim to improve the cytotoxic potential by utilizing the human effector domains Granzyme B and MAP-tau. Specific binding to the target cells as well as functionality of the Granzyme B effector domain were shown, enabling us to directly compare the constructs with the respective original immunotoxins. Furthermore, we were able to establish a s.c. tumor in SCID mice using cells with a high sensitivity to the used immunotoxin, which exhibits a potent cytotoxicity in the nanomolar range. Determination of the maximal tolerable dose of the immunotoxin is ongoing, so far no detrimental effects were observed. Take home message: SNAP-tag labeled antibody fragments and cytolytic fusion proteins show highly specific binding and killing of leukemic cells and thus are excellent candidates for reliable diagnosis and targeted therapy of leukemia. Their optimization regarding internalization behavior, cytotoxic potency and human immunocompatibility is currently addressed.

PURPOSE: Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA' against EGFR(+) RMS cells in vitro and ex vivo. METHODS: We tested the specific binding and internalization behavior of 425(scFv)-ETA' in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA' was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients. RESULTS: We confirmed the specific binding of 425(scFv)-ETA' to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR(+) RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line. CONCLUSIONS: This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA'-based IT on RMS cells. We confirmed the selective killing with IC50 values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material.

29th European Photovoltaic Solar Energy Conference and Exhibition; 976-979, This paper presents an up-scalable solution for the formation of doped areas based on inkjet printing of boron and phosphorous doping inks. The influence of the thickness of the inkjet-printed doping source layers and the diffusion temperature on the sheet resistance is evaluated. In addition, the doping profiles are measured. For a diffusion temperature of 950 °C a sheet resistance of 50 Ω/sq is achieved with the boron ink. At the same temperature a sheet resistance of 18 Ω/sq is achieved with the boron ink. The process is then for the first time applied for the fabrication of back-contact back-junction solar cells with inkjet-printed p+-emitter. The best solar cell features an efficiency of = 20.6 %. To date, these are the best solar cells with an inkjet-printed doping source. Lifetime samples that are processed in parallel exhibit an emitter saturation current density below 90 fA/cm2.

Targeted therapies for the treatment of cancer, but also inflammation and autoimmune diseases will reduce major side effects accompanied with conventional treatment modalities. The immunotoxin concept uses bacterial or plant toxins, coupled to antibodies or natural ligands targeting cancer cells. Initially, immunotoxins suffered from drawbacks like nonspecific cytotoxicity. Even the third generation of immunotoxins comprised of truncated antibodies and modified effector molecules experienced clinical set-backs due to immune responses. Long-term treatment of cancer and non-life-threatening chronic inflammatory diseases requires their complete 'humanization'. This lead to evaluating human cytolytic fusion proteins (hCFPs), based on human apoptosis-inducing proteins. Lacking an endogenous translocation domain dramatically reduces the cell-death inducing capacity of such proteins. Here, we report on optimizing hCFPs, based on the anti-CD64 single chain variable fragment H22(scFv), specifically eliminating CD64+ macrophages and malignant progenitor cells. We replaced the bacterial toxin in H22(scFv)-ETA' with the pro-apoptotic human granzyme B or angiogenin. Translocation was promoted by a sophisticated adapter containing a membrane transfer peptide (MTD) flanked by endosomal and cytosolic cleavable peptides, thus achieving in vitro cytotoxic activity comparable to bacterial immunotoxins. We demonstrate for the first time that optimized hCFPs, based on granzyme B or angiogenin, can compete with classical ETA-based immunotoxins.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays a critical role in many inflammatory diseases. Soluble TNF can be neutralized by monoclonal antibodies (mAbs), and this is a widely-used therapeutic approach. However, some patients do not respond to anti-TNF therapy due to the increased expression of CD64 on monocytes and macrophages. A recent study has shown that CD64 captures anti-TNF mAbs via their Fcg domain, which induces the transcription of pro-inflammatory genes. Specific blocking of CD64 could therefore be a promising strategy to improve the response to anti-TNF therapy. We used the CD64-specific antibody fragment H22(scFv) and tested its activity against the human CD64+ cell line HL-60. When stimulated with interferon gamma (IFN-g), these cells represent a pro-inflammatory phenotype of the monocyte/macrophage lineage. We found that H22(scFv) binds selectively to and blocks CD64, preventing the capture of anti-TNF mAb. Importantly, H22(scFv) itself does not induce CD64 activation. We also found that transmembrane TNF on HL-60 cells stimulated with IFN-g also contributes to the capture of anti-TNF mAb, although via their Fab domain. In conclusion, the specific blocking of CD64 by H22(scFv) could be used a possible anti-inflammatory mechanism for potentiating the effect of anti-TNF antibodies.

Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) are rare lymphoproliferative cancer types. Although most HL patients can be cured by chemo- and radio-therapy, 4-50% of patients relapse and have a poor prognosis. The need for improved therapeutic options for patients with relapsed or refractory disease has been addressed by CD30-specific antibody-based immunotherapeutics. However, available CD30-specific monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) or chimeric immunotoxins suffer from the requirement of a functional host immunity, undesirable immune reactions or heterogeneity and instability, respectively. Here, we present a new fusion protein comprised of the CD30-specific antibody single-chain fragment Ki4(scFv) and the human pro-apoptotic effector protein, microtubule-associated protein tau (MAPT). Ki4(scFv)-MAP selectively induced apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner. Tubulin polymerization assays confirmed that Ki4(scFv)-MAP stabilizes microtubules, suggesting a mechanism for its pro-apoptotic action. Dose-finding experiments proved that Ki4(scFv)-MAP is well tolerated in mice compared to the previously reported Ki4(scFv)-ETA'. Ki4(scFv)-MAP significantly inhibited growth of subcutaneous L540cy xenograft tumours in mice. Our data present a novel approach for the treatment of CD30+ lymphomas, combining the binding specificity of a target-specific antibody fragment with the selective cytotoxicity of MAPT towards proliferating lymphoma cells.

Substrates for plasmonic sensors in a flow-through configuration are mostly fabricated by cost-intensive clean room processes, whereas high-volume diagnostic devices are typically made of polymers. This contrast could limit the application of this efficient flow regime in mass-produced devices. In order to become more compatible with polymer processing, a commercially available polycarbonate filter membrane has been evaluated as a substrate for plasmonic flow-through biosensing. The membrane has been sputtered with gold and its sensitivity to changes of bulk refractive index has been determined by transmission measurements using sodium chloride solutions. The sensitivity has been evaluated by determining the wavelength barycenter in a wavelength interval from 470 to 800 nm. The highest determined sensitivity to variations in bulk refractive index is 117 nm RIU-1 (refractive index units). This sensitivity is smaller than that of regular arrays of nanoholes. But the integrating character of the applied evaluation leads to an average standard deviation of 0.005 nm which results in a resolution of 4.1 10 -5 RIU. This resolution is sufficient for the detection of protein adsorptions. The proof of principle has been shown with bovine serum albumin and a simplified immunoassay, which consists of the sequential addition of protein A, an IgG antibody and its corresponding antigen. The results show the applicability of this polymeric membrane for biosensing applications. These substrates could enable plasmonic sensing in a flow-through configuration in disposable diagnostic devices.

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.

28th European Photovoltaic Solar Energy Conference and Exhibition; 1131-1134, Up to now most spin-on diffusion sources based on a wet chemically created boron-silicate glass (BSG) lead to a strongly degraded carrier lifetime after diffusion. In this study a spin-on boron diffusion source is applied to the fabrication of large area n-type CZ bifacial solar cells and the performance of the spin-on source is compared to a BBr3 boron diffusion concerning the resulting lifetime and emitter saturation current J0e, shunting behaviour and external quantum efficiency. Comparable J0e values and a comparable lifetime were achieved resulting in a top efficiency of 19.38% in comparison to a top efficiency of 19.60% achieved by BBr3 diffusion. Further investigation of the electrical parameters showed the possibility of improvement in cell performance.

Background: Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs). Methods: Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging. Results: The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model. Conclusion: Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.

Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.