Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting.

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

Affinity maturation by semi-rational approaches.

Rational engineering methods can be applied with success to optimize physicochemical characteristics of antibodies. Application of in silico analysis and prediction methods to antibody Fv regions can help to find residues affecting antibody-antigen affinity when high-resolution antibody structures or antibody-antigen complex structures are known. In these cases, the identification of residues affecting affinity can facilitate the selection of candidates for guided maturation by PCR using degenerate oligonucleotides. Here, we describe the utilization of a semi-rational approach to enhance the affinity of antibodies by combining in silico and traditional wet lab-based methods.

Design, synthesis and evaluation of high-affinity binders for the celiac disease associated HLA-DQ2 molecule.

Celiac disease is caused by uncontrolled CD4 T-cell responses directed to wheat-derived gluten peptides bound to the disease predisposing HLA-DQ molecules. The only available treatment is a life-long gluten-free diet which is complicated by the widespread use of wheat-derived gluten in the food industry. As the binding of gluten-derived peptides is a prerequisite for the induction of the inflammatory T-cell response, blockers that would prevent gluten peptide binding to the HLA-DQ molecules might be used as an alternative to the gluten-free diet. In the present study we have analyzed the binding properties of a set of previously identified natural ligands for HLA-DQ2, the primary disease predisposing allele. An in silico method, Epibase, ranked these peptides and the top one, a peptide with a nine amino acid core FVAEYEPVL, was measured among these peptides as the peptide with the highest binding affinity for HLA-DQ2. In a stepwise approach we subsequently tested the impact of N-terminal extensions and systematic single amino acid substitutions within the core of this peptide which revealed that an N-terminal extension with the tripeptide sequence ADA increased binding affinity 5- to 6-fold. In addition the substitution analysis indicated which amino acids were most preferred at anchor residues in the lead peptide, generally leading to an increase of binding affinity with a factor of 2. Next we tested which combinations of such preferred amino acids yielded the best results. The combined results indicate that a peptide with sequence ADAYDYESEELFAA (core in bold) had superior binding properties. This peptide was chosen as a lead peptide for further optimization with non-natural amino acids at the p1 position, since molecular modeling indicated that none of the natural amino acids is able to optimally occupy the p1 pocket. A set of 8 non-proteinogenic amino acids was designed, synthesized and incorporated in the lead peptide (and in two control peptides) and tested for binding to HLA-DQ2. The results indicate that the effect of the incorporation of these non-proteinogenic amino acids depended on the peptide in which they were incorporated and that the maximum increase in binding affinity obtained was approximately 2-fold. Altogether lead sequences were obtained that have a binding affinity for HLA-DQ2 that is 100- to 200-fold higher compared to that of the gluten-derived peptide that has the highest affinity for HLA-DQ2. Such peptides are candidate lead peptides for further optimization. Our results, however, also indicate that in order to obtain further significant increases in binding affinity alternative approaches will have to be explored.

Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.

BACKGROUND: The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. METHODS AND FINDINGS: Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM(+) memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. CONCLUSIONS: The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM(+) memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.

Nitric oxide- and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are regulatory molecules in various developmental processes and stress responses. Tobacco (Nicotiana tabacum) leaves exposed to moderate high light dramatically potentiated NO-mediated cell death in catalase-deficient (CAT1AS) but not in wild-type plants, providing genetic evidence for a partnership between NO and H(2)O(2) during the induction of programmed cell death. With this experimental model system, the specific impact on gene expression was characterized by either NO or H(2)O(2) alone or both molecules combined. By means of genome-wide cDNA-amplified fragment length polymorphism analysis, transcriptional changes were compared in high light-treated CAT1AS and wild-type leaves treated with or without the NO donor sodium nitroprusside. Differential gene expression was detected for 214 of the approximately 8,000 transcript fragments examined. For 108 fragments, sequence analysis revealed homology to genes with a role in signal transduction, defense response, hormone interplay, proteolysis, transport, and metabolism. Surprisingly, only 16 genes were specifically induced by the combined action of NO and H(2)O(2), whereas the majority were regulated by either of them alone. At least seven transcription factors were mutually up-regulated, indicating significant overlap between NO and H(2)O(2) signaling pathways. These results consolidate significant cross-talk between NO and H(2)O(2), provide new insight into the early transcriptional response of plants to increased NO and H(2)O(2) levels, and identify target genes of the combined action of NO and H(2)O(2) during the induction of plant cell death.

Benchmarking the CATMA microarray. A novel tool for Arabidopsis transcriptome analysis.

Transcript profiling is crucial to study biological systems, and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the performance of the CATMA microarray designed for Arabidopsis (Arabidopsis thaliana) transcriptome analysis and compared it with the Agilent and Affymetrix commercial platforms. The CATMA array consists of gene-specific sequence tags of 150 to 500 bp, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (The Institute for Genomic Research release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled targets derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. CATMA arrays showed the largest dynamic range extending over three to four logs. Agilent and Affymetrix arrays displayed a narrower range, presumably because signal saturation occurred for transcripts at concentrations beyond 1,000 copies per cell. Sensitivity was comparable for all three platforms. For Affymetrix GeneChip data, the RMA software package outperformed Microarray Suite 5.0 for all investigated criteria, confirming that the information provided by the mismatch oligonucleotides has no added value. In addition, taking advantage of replicates in our dataset, we conducted a robust statistical analysis of the platform propensity to yield false positive and false negative differentially expressed genes, and all gave satisfactory results. The results establish the CATMA array as a mature alternative to the Affymetrix and Agilent platforms.

Transcript profiling of early lateral root initiation.

At the onset of lateral root initiation in Arabidopsis thaliana, the phytohormone auxin activates xylem pole pericycle cells for asymmetric cell division. However, the molecular events leading from auxin to lateral root initiation are poorly understood, in part because the few responsive cells in the process are embedded in the root and are thus difficult to access. A lateral root induction system, in which most xylem pole pericycle cells were synchronously activated by auxin transport inhibition followed by auxin application, was used for microarray transcript profiling. Of 4,600 genes analyzed, 906 significantly differentially regulated genes were identified that could be grouped into six major clusters. Basically, three major patterns were discerned representing induced, repressed, and transiently expressed genes. Analysis of the coregulated genes, which were specific for each time point, provided new insight into the molecular regulation and signal transduction preceding lateral root initiation in Arabidopsis. The reproducible expression profiles during a time course allowed us to define four stages that precede the cell division in the pericycle. These early stages were characterized by G1 cell cycle block, auxin perception, and signal transduction, followed by progression over G1/S transition and G2/M transition. All these processes took place within 6 h after transfer from N-1-naphthylphthalamic acid to 1-naphthalene acetic acid. These results indicate that this lateral root induction system represents a unique synchronized system that allows the systematic study of the developmental program upstream of the cell cycle activation during lateral root initiation.