Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

In this case study, we describe the use of in vitro protein evolution with ribosome display to improve the potency of a human interleukin-13-neutralising antibody by a factor of over 200-fold and derive a therapeutic candidate, CAT-354, for the treatment of asthma. A combination of directed and random mutagenesis enabled the identification of highly potent neutralising antibodies and highlighted the advantage of the ribosome display protein evolution approach in identifying beneficial mutations across the entire sequence space. This chapter describes in detail the process followed to achieve a successful in vitro affinity maturation outcome using ribosome display technology.

Intracellular Ribosome Display Via SecM Translation Arrest as a Selection for Antibodies with Enhanced Cytosolic Stability

Ribosome display presents an innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Displayed proteins are bound to and recovered from target molecules in multiple rounds of selection in order to enrich for specific binding proteins. No transformation step is necessary, which could lead to a loss of library diversity. A cycle of display and selection can be performed in one day, enabling the existing gene repertoire to be rapidly scanned. Proteins isolated from the panning rounds can be further modified through random or directed molecular evolution for affinity maturation, as well as selected for characteristics such as protein stability, folding and functional activity. Recently, the field of display technologies has become more prominent due to the generation of new scaffolds for ribosome display, isolation of high-affinity human antibodies by phage display, and their implementation in the discovery of novel protein–protein interactions. Applications for this technology extend into the broad field of antibody engineering, proteomics, and synthetic enzymes for diagnostics and therapeutics in cancer, autoimmune and infectious diseases, neurodegenerative diseases and inflammatory disorders. This review highlights the role of ribosome display in drug discovery, discusses advantages and disadvantages of the system, and attempts to predict the future impact of ribosome display technology on the development of novel engineered biopharmaceutical products for biological therapies.

Basidiomycete Lentinula edodes CDC5 and a novel interacting protein CIPB bind to a newly isolated target gene in an unusual manner

A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.

In crayfish claw closer muscle, the giant sarcomeres are 8.3 microm long at rest, four times longer than vertebrate striated muscle sarcomeres, and they are extensible up to 13 microm upon stretch. Invertebrate connectin (I-connectin) is an elastic protein which holds the A band at the center of the sarcomere. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53 424 bp (17 352 residues; 1960 kDa). Crayfish I-connectin contains two novel 68- and 71-residue repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the 68-residue repeats revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions rich in charged amino acids, occupying 63% of the crayfish I-connectin molecule, may allow a span of a 3.5 microm distance as a new class of composite spring.

Vif and Vpr are HIV-1 accessory proteins that create optimal conditions for viral replication. They are considered as potential targets for the development of therapeutic agents. Natural amino acid substitutions in these proteins have previously been associated with disease progression. The aim of this study was to analyze the genetic diversity of Vif and Vpr in HIV-1 group M clades. A total of 5286 sequences were downloaded and analyzed. For 37 clades in group M, the consensus sequences, amino acid natural variation, and clade-specific amino acid residue substitutions (CSSs) were evaluated. Structural analysis and modeling of consensus sequences were performed for subtypes A1, B, C, and D. The average conservation degree in the HIV-1 group M was 86.4% for Vif and 91.3% for Vpr. In both proteins, the lowest amino acid diversity was observed in sub-subtype A6, and the highest in subtype B. In consensus sequences, the substitutions, which might influence pathogenesis, have been determined: in Vif—22H (11_cpx, 91_cpx) and 136P (A6, 01_AE, 15_01B, 59_01B, 89_BF1, 103_01B, 111_01C, 133_A6B), in Vpr—41N (06_cpx) and 55A (B, 07_BC, 35_01D, 56_cpx, 66_cpx, 66_BF1, 71_BF1, 85_BC, 137_0107). In functional motifs, CSSs associated with changes in the chemical properties of amino acid residues were noted. These findings could be taken into account for the development of therapeutic drugs in the future. No correlation was observed between the subtypes and the spatial organization of the oligomeric structures of Vif and Vpr. Using the structural analysis and modeling, it has been shown for the first time that Vif can interact with APOBEC3G as an oligomer.

Two general solid-phase methods for the synthesis of a new class of 2,5-diketopiperazines (DKPs) containing the trans-4-hydroxy-L-proline amino acid residue (Hyp) have been developed. An N-protected hydroxyproline methyl ester was linked through the hydroxyl function to the Ellman resin. The synthesis procedures were conceived to enable a sequence of Hyp alkylation, Hyp N-acylation, cyclization, and amide bond alkylation. Up to three different centers of molecular diversity were introduced around the DKP scaffold. Highly functionalized bicyclic compounds were obtained in good yield and purity. The alkylation of hydroxyproline alpha CH was performed without control of the diastereoselectivity. During the final alkylation of the backbone, amide bond epimerization at the alpha-carbon atoms of the two amino acid residues was observed. The structures of representative DKPs were elucidated with multidimensional NMR experiments. The described reaction pathways can be applied to the identification of heterocyclic molecule inhibitors to diverse enzyme targets.

The hydrophobic domains in the carboxyl-terminal signal for GPI modification and in the amino-terminal leader peptide have similar structural requirements

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.

Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross-reactivity against widely divergent circulating strains. Computer-generated sequences minimize genetic distance between circulating strains, and can be generated as a consensus sequence (most common amino acid at each position) or a representative sequence (derived with Bayesian and maximal likelihood phylogenetic tools). Broad recognition of such sequences in HCV has not been demonstrated. Consensus and representative sequences were generated from 390 full-length HCV genotype 1a polypeptide sequences. Sequence mutations in known epitopes were identified by longitudinal sequencing of HCV-infected subjects. Peptides encoding consensus, representative, and variant epitope sequences were tested for the capacity to expand CD8 T cells from HCV-infected subjects and to elicit cross-reactive responses by IFNg ELISpot. The representative and consensus sequences were identical for 9/11 epitopes examined. T cell lines generated against representative sequence peptides were generally cross-reactive to consensus sequence and natural sequence variants. Furthermore, representative sequence peptides generated more robust T cell responses than did natural sequence variant peptides. The broadest recognition of highly diverse circulating HCV strains was achieved using CD8+ T cells expanded with representative sequence HCV. These data support the use of representative sequence in HCV vaccine design.

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.

Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds

The gene for the common alpha subunit of the porcine anterior pituitary glycoprotein hormones was cloned from a genomic library constructed in EMBL3. The nucleotide sequence of the entire coding sequence of the porcine common alpha-subunit gene was determined in addition to one intron and 1059 and 160 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the common alpha-subunit gene is present as a single copy. The transcriptional unit of the porcine common alpha subunit spanned about 14 kb and contained four exons interrupted by three introns of about 11.5, 1.2 and 0.4 kb. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues +9/+10 and +71/+72 were highly conserved among the rat, human and bovine common alpha-subunit genes. In the proximal portion of the 5'-flanking region, one TATA box and one CCAAT box were present. A steroid-responsive element was not found up to 1059 bases upstream from the transcription start site. The potential AP-1 and AP-2 factor-responsive elements were present at three and one positions respectively in the 5'-flanking region. This feature suggests that hypothalamic gonadotrophin-releasing hormone stimulates the expression of the common alpha-subunit gene predominantly by a signal-transduction system, with the protein kinase C cascade and factors AP-1 and AP-2 as mediators. The cyclic AMP-responsive element was also present at two positions, but a single base substitution was found in each sequence compared with the consensus sequence.(ABSTRACT TRUNCATED AT 250 WORDS)