Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.     

The population genetics of Quechuas, the largest native South American group: autosomal sequences, SNPs, and microsatellites evidence high level of diversity

Elucidating the pattern of genetic diversity for non-European populations is necessary to make the benefits of human genetics research available to individuals from these groups. In the era of large human genomic initiatives, Native American populations have been neglected, in particular, the Quechua, the largest South Amerindian group settled along the Andes. We characterized the genetic diversity of a Quechua population in a global setting, using autosomal noncoding sequences (nine unlinked loci for a total of 16 kb), 351 unlinked SNPs and 678 microsatellites and tested predictions of the model of the evolution of Native Americans proposed by (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496). European admixture is <5% and African ancestry is barely detectable in the studied population. The largest genetic distances were between African versus Quechua or Melanesian populations, which is concordant with the African origin of modern humans and the fact that South America was the last part of the world to be peopled. The diversity in the Quechua population is comparable with that of Eurasian populations, and the allele frequency spectrum based on resequencing data does not reflect a reduction in the proportion of rare alleles. Thus, the Quechua population is a large reservoir of common and rare genetic variants of South Amerindians. These results are consistent with and complement our evolutionary model of South Amerindians (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496), proposed based on Y-chromosome data, which predicts high genomic diversity due to the high level of gene flow between Andean populations and their long-term effective population size.     

Methanogenic activities in alpine soils

Uncontrolled microbial methane production is playing an important role in global warming. In the present study, we showed that water content and incubation temperature increase the potential for methane formation in the two alpine soils under investigation. Beside these factors, the grazing of cows and thus the amendment of methanogenic microorganisms by cattle dung is the most important factor determining the potential of methane production in those soils.     

TLR and nucleotide-binding oligomerization domain-like receptor signals differentially regulate exogenous antigen presentation

The effect of dendritic cell (DC) maturation on MHC class II-restricted Ag presentation is well studied, but less is known about the effects of DC maturation on MHC class I-restricted cross-presentation. We investigated the ability of mature DCs to present Ags from cells infected with HSV-1. Pretreatment with pure LPS increased cross-presentation in a manner dependent on both MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-β, whereas a similar dose of a less pure LPS preparation inhibited cross-presentation. The difference could not be attributed to differences in uptake or phenotypic maturation. The likely contaminant responsible for shutting down cross-presentation is peptidoglycan (PGN). Addition of PGN to pure LPS abrogated its ability to enhance cross-presentation. Direct activation of DCs with PGN inhibited cross-presentation through nucleotide-binding oligomerization domain-like receptor signaling. These results demonstrate that different maturation stimuli can have opposite impacts on the ability of DCs to cross-present viral Ags.     

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.     

Diphenyl ditelluride targets brain selenoproteins in vivo: inhibition of cerebral thioredoxin reductase and glutathione peroxidase in mice after acute exposure

In this study, we investigated the effect of diphenyl ditelluride (PhTe)₂ administration (10 and 50?μmol/kg) on adult mouse behavioral performance as well as several parameters of oxidative stress in the brain and liver. Adult mice were injected with (PhTe)₂ or canola oil subcutaneously (s.c.) daily for 7?days. Results demonstrated that (PhTe)₂ induced prominent signs of toxicity (body weight loss), behavioral alterations and increased in lipid peroxidation in brain. 50?μmol/kg (PhTe)₂ inhibited blood δ-aminolevulinic acid dehydratase (δ-ALA-D), a redox sensitive enzyme. (PhTe)₂ caused an increase in cerebral non-protein thiol (NPSH) and protein thiol (PSH) groups. In the liver, 50?μmol/kg (PhTe)₂ decreased NPSH, but did not alter the content of protein thiol groups. (PhTe)₂ decreased cerebral antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), and thioredoxin reductase (TrxR). In liver, (PhTe)₂ increase SOD and GR and decreased GPx activity. Results obtained herein suggest that the brain was more susceptible to oxidative stress induced by (PhTe)₂ than the liver. Furthermore, we have demonstrated for the first time that TrxR is an in vivo target for (PhTe)_2. Combined, these results highlight a novel molecular mechanism involved in the toxicity of (PhTe)₂. In particular the inhibition of important selenoenzymes (TrxR and GPx) seems to be involved in the neurotoxicity associated with (PhTe)₂ exposure in adult mice.     

Induction of Cell Cycle Arrest and Apoptosis by Ruthenium Complex cis-(Dichloro)tetramineruthenium(III) Chloride in Human Lung Carcinoma Cells A549

Lung cancer is one of the leading causes of death in the world, and non-small cell lung carcinoma (NSCLC) accounts for approximately 75-85% of all lung cancers. In the present work, we studied the cytotoxic activity, cell cycle arrest and induction apoptosis of the compound cis-(dichloro)tetramineruthenium(III) chloride {cis-[RuCl(2)(NH(3))(4)]Cl} in human lung carcinoma tumor cell line A549. The results of MTT and trypan blue assays showed that cis-[RuCl(2)(NH(3))(4)]Cl causes reduction in the viability of A549 cells when treating with 95 and 383 μM of the compound for 48 and 72 h. Lower concentrations of the compound (19, 3.8 and 0.38 μM), however, only slightly affected cell viability. The IC(50) value for the compound was about 383 μM. Survival analysis of the A549 cells after treatment with ruthenium(III) compound using long term clonogenic assay showed that it reduced colony formation ability at concentrations of 0.38 and 3.8 μM, and at concentrations of 95 and 383 μM no colonies were observed. Cell cycle analysis showed that compound ruthenium led to an accumulation of A549 cells in S phase and increased in the sub-G1 peak. In addition, cis-(dichloro)tetramineruthenium(III) chloride treatment induced apoptosis, as observed by the increased numbers of annexin V-positive cells and increased messenger RNA expression of caspase-3.     

A hormonal misunderstanding in Acca sellowiana embryogenesis: levels of zygotic embryogenesis do not match those of somatic embryogenesis

Endogenous levels of IAA, ABA and four types of CKs were analyzed in zygotic and indirect (ISE) and direct somatic embryogenesis of Acca sellowiana. Zygotic and somatic embryos at different developmental stages were sampled for morphological and hormonal analysis. Both embryo types showed substantial asymmetry in hormone levels. Zygotic embryos displayed a conspicuous peak of IAA in early developmental stages. The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes. The short transient pulse of IAA observed on the 3rd day in culture was suggested to be involved with the signaling for the induction of somatic embryogenesis. Fertilized ovule development was associated with increased IAA levels 21?C24?days after pollination, followed by a sharp decrease in the cotyledonary stage, both in zygotic and somatic embryos. There was a prominent increase in ABA levels in cultures which generated ISE 24?C30?days after pollination, a period that corresponds to the heart and torpedo stages. The levels of total CKs (Z, [9R]Z, iP and [9R]iP) were also always higher in zygotic than in somatic embryogenesis. While zygotic embryogenesis was dominated by the presence of zeatin, the somatic process, contrarily, was characterized by a large variation of the other cytokinin forms and amounts studied. The above results, when taken together, could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.