Phage Display-Based Nanotechnology Applications in Cancer Immunotherapy.

Phage display is a nanotechnology with limitless potential, first developed in 1985 and still awaiting to reach its peak. Awarded in 2018 with the Nobel Prize for Chemistry, the method allows the isolation of high-affinity ligands for diverse substrates, ranging from recombinant proteins to cells, organs, even whole organisms. Personalized therapeutic approaches, particularly in oncology, depend on the identification of new, unique, and functional targets that phage display, through its various declinations, can certainly provide. A fast-evolving branch in cancer research, immunotherapy is now experiencing a second youth after being overlooked for years; indeed, many reports support the concept of immunotherapy as the only non-surgical cure for cancer, at least in some settings. In this review, we describe literature reports on the application of peptide phage display to cancer immunotherapy. In particular, we discuss three main outcomes of this procedure: (i) phage display-derived peptides that mimic cancer antigens (mimotopes) and (ii) antigen-carrying phage particles, both as prophylactic and/or therapeutic vaccines, and (iii) phage display-derived peptides as small-molecule effectors of immune cell functions. Preclinical studies demonstrate the efficacy and vast potential of these nanosized tools, and their clinical application is on the way.

Assessment of antibody library diversity through next generation sequencing and technical error compensation.

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.

Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.

The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells.

BACKGROUND: Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5' UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3. RESULTS: In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10-15 days after last administration of 5-azadC. CONCLUSIONS: Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene.

Defining the role of the CGGBP1 protein in FMR1 gene expression.

Fragile X syndrome is the most common heritable form of intellectual disability and is caused by the expansion over 200 repeats and subsequent methylation of the CGG triplets at the 5' UTR of the FMR1 gene, leading to its silencing. The epigenetic and molecular mechanisms responsible for FMR1 gene silencing are not fully clarified. To identify structure-specific proteins that could recruit components of the silencing machinery we investigated the role of CGGBP1 in FMR1 gene transcription. CGGBP1 is a highly conserved protein that binds specifically to unmethylated CGG tracts. Its role on FMR1 transcription is yet to be defined. Sequencing analysis and expression studies through quantitative PCR of CGGBP1 were performed in cell lines with different allele expansions: wild type, premutation, methylated full mutation and unmethylated full mutation, demonstrating no differences between them. ChIP assays clearly demonstrated that CGGBP1 binds to unmethylated CGG triplets of the FMR1 gene, but not to methylated CGGs. We also observed that CGGBP1 binding to the FMR1 locus was restored after pharmacological demethylation, with 5-azadC, of alleles, carriers of methylated full mutation, suggesting a possible role for CGGBP1 in FMR1 expression. CGGBP1 silencing with shRNAs (reaching ~98% of CGGBP1-mRNA depletion) did not affect FMR1 transcription and CGG expansion stability in expanded alleles. Although the strong binding to the CGG tract could suggest a relevant role of CGGBP1 on FMR1 gene expression, our results demonstrate that CGGBP1 has no direct effect on FMR1 transcription and CGG repeat stability.

Role of CTCF protein in regulating FMR1 locus transcription.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.

Rhodium and iridium salts inhibit proliferation and induce DNA damage in rat fibroblasts in vitro.

Environmental concentration of the platinum group elements is increased in the last years due to their use in automobile catalytic converters. Limited data are available on the effects of such elements at a cellular level and on their toxicity, especially for rhodium and iridium which have been more recently introduced in use. The toxic effects of rhodium and iridium salts were analyzed on a normal diploid rat fibroblast cell line in vitro. Both salts halted cell growth in a dose- and time-dependent fashion by inhibiting cell cycle progression, inducing apoptosis and modulating the expression of cell cycle regulatory proteins. In fact, they both caused an accumulation of cells in the G2/M phase of the cell cycle and affected the expression levels of pRb, cyclins D1 and E, p21(Waf1) and p27(Kip1). DNA strand breaks, as assessed by comet test, and an increase in the intracellular levels of reactive oxygen species also occurred in exposed cell cultures. These findings suggest a potential toxicity of both iridium and rhodium salts and emphasize the need for further studies to understand their effects at a cellular level to enable a better assessment of their toxic effects and to identify ways for their modulation and/or prevention.

Evaluation of in vitro toxic effects of cement dusts: a preliminary study.

Cement is widely used for construction and several reports have suggested a potential toxicity of cement dusts although it has never been definitively assessed. To determine the cytotoxic and bioactive effects of cement dusts, cultures of normal rat fibroblasts were exposed to different types of cements and cell growth parameters, apoptosis and the occurrence of DNA damage (both in terms of DNA breaks and oxidative damage) were analyzed. Cells were exposed to cement extracts or cultured in direct contact with cement dusts and the results obtained were compared to cells cultured in fresh medium. A dose-dependent decrease in viable cells was observed with all tested cements. Different results were obtained in the cell-cement direct contact tests compared to the indirect contact tests performed using extracts. Inhibition of cell growth was associated in most cases with an accumulation of cells in the S-phase of the cell-cycle and the appearance of an apoptotic peak. DNA strand breaks, assessed by comet test, and increase in the levels of 8-OHdG, an important marker of DNA oxidative damage, always occurred by incubating cells in the presence of cement extracts or dusts. However, after removal of cement, a rapid damage repair was generally observed with an almost complete recovery within 12 hours. In conclusion, all cements analyzed in this study displayed a limited toxicity in vitro without significant differences amongst them. Overall, the results obtained indicate that cements should be treated as hazardous materials but they do not allow to make accurate predictions regarding the in vivo effects. Further studies are warranted to reach a better understanding of the potential toxic effects of cements, to identify the responsible mechanisms and to evaluate the possibility of modulating and/ or preventing them.

Identification of Sp1 and GC-boxes as transcriptional regulators of mouse Dag1 gene promoter.

Dystroglycan is a widely expressed adhesion complex that anchors cells to the basement membrane and is involved in embryonic development and differentiation. Dystroglycan expression is frequently reduced in human dystrophies and malignancies, and its molecular functions are not completely understood. Several posttranslational mechanisms have been identified that regulate dystroglycan expression and/or function, while little is known about how expression of the corresponding Dag1 gene is regulated. This study aimed to clone the Dag1 gene promoter and to characterize its regulatory elements. Analysis of the mouse Dag1 gene 5'-flanking region revealed a TATA and CAAT box-lacking promoter including a GC-rich region. Transfection studies with serially deleted promoter constructs allowed us to identify a minimal promoter region containing three Specificity protein 1 (Sp1) sites and an E-box. Sp1 binding was confirmed by chromatin immunoprecipitation assay, and Sp1 downregulation reduced dystroglycan expression in muscle cells. Treatment with 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A increased Dag1 mRNA expression levels in myoblasts, and methylation decreased promoter activity in vitro. Furthermore, Dag1 gene promoter methylation was reduced while its expression increased during differentiation of C(2)C(12) myoblast cells in myotubes. In conclusion, for the first time we have characterized the activity of the mouse Dag1 gene promoter, confirming a complex regulation by Sp1 transcription factor, DNA methylation, and histone acetylation, which might be relevant for a better understanding of the physiopathology of the dystroglycan complex.