Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.     

Irrelevance of Microsatellite Instability in the Epidemiology of Sporadic Pancreatic Ductal Adenocarcinoma

Background and Aims

Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.

Methods

MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.

Results

Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.

Conclusions

MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.     

Genetic variation in cell death genes and risk of non-Hodgkin lymphoma

Background

Non-Hodgkin lymphomas are a heterogeneous group of solid tumours that constitute the 5^th highest cause of cancer mortality in the United States and Canada. Poor control of cell death in lymphocytes can lead to autoimmune disease or cancer, making genes involved in programmed cell death of lymphocytes logical candidate genes for lymphoma susceptibility.

Materials and Methods

We tested for genetic association with NHL and NHL subtypes, of SNPs in lymphocyte cell death genes using an established population-based study. 17 candidate genes were chosen based on biological function, with 123 SNPs tested. These included tagSNPs from HapMap and novel SNPs discovered by re-sequencing 47 cases in genes for which SNP representation was judged to be low. The main analysis, which estimated odds ratios by fitting data to an additive logistic regression model, used European ancestry samples that passed quality control measures (569 cases and 547 controls). A two-tiered approach for multiple testing correction was used: correction for number of tests within each gene by permutation-based methodology, followed by correction for the number of genes tested using the false discovery rate.

Results

Variant rs928883, near miR-155, showed an association (OR per A-allele: 2.80 [95% CI: 1.63–4.82]; p_F = 0.027) with marginal zone lymphoma that is significant after correction for multiple testing.

Conclusions

This is the first reported association between a germline polymorphism at a miRNA locus and lymphoma.     

The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.     

Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1: COMPARISON OF DARPin AND CAMELID VHH BINDING MODE*

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry for the production of an array of fermented milk products. Several industrial strains are sensitive to various distinct bacteriophages, mostly belonging to the Siphoviridae family. The lactococcal phage population is divided in at least 10 genetically distinct groups, of which the 936, c2, and P335 groups are prominent (1, 2). These L. lactis-infecting phages are considerably problematic in causing milk fermentation failures and resulting in decreased yields as well as low quality products (3). Preventing these infections has proven to be difficult because of lactococcal phage ubiquity, biodiversity, and genomic plasticity (4).Phage infection is initiated by binding of the phage receptor-binding protein (RBP),⁵ located within the baseplate at the distal part of the tail, to its receptor on the host cell surface (5). We have previously solved the crystal structures of the three RBPs of the lactococcal phages p2 (936) (6), bIL170 (936) (7), TP901-1 (P335) (8), and their chimera (9) as well as characterized their saccharide binding sites (10). The RBPs of these phages have a similar homotrimeric architecture related by a 3-fold axis. They comprise three domains: the N terminus shoulder domain, the interlaced β-prism neck domain, and the jellyroll head domain at the C terminus. The head domain has a saccharide binding site likely involved in host recognition. The lactococcal phage TP901-1 contains a double-disk-shaped baseplate at the tip of its tail which is made of a lower baseplate protein (BppL) and an upper baseplate protein (BppU) (11).One strategy to minimize bacteriophage infections is to competitively block phage adsorption by adding a protein that specifically binds to the phage RBP. A neutralizing llama VHH domain recognizing the head domain of the phage p2 RBP has been used to block L. lactis phage infection in milk fermentation (12). Lactococcal phages could readily escape neutralization by generating mutations interfering with VHH binding over the large interaction surface while keeping the central polysaccharide receptor binding pocket intact (10). Designed ankyrin repeat proteins (DARPins) may be another tool to neutralize viral infection, as they display distinct characteristics from VHHs and contain the required properties in terms of stability and facility of expression (13).Ankyrin repeat proteins are found in virtually all phyla and mediate specific protein-protein interactions in all cell compartments (14). The ankyrin elementary module is composed of 33 amino acids structured as a β-turn followed by two antiparallel α-helices and a loop connected to the β-turn of the next repeat. The repeats are stacked in a rigid manner. In creating a DARPin library, residues in each repeat were subdivided in two groups; (i) randomized residues constituting potential target interaction points and (ii) framework residues, important for maintaining the ankyrin fold (13). Libraries with varying repeat numbers were assembled and named according to the constituent repeat number; N2C and N3C libraries were used in this study, with two and three internal repeats inserted between the N and C capping repeats, respectively. DARPins are a powerful alternative to the use of antibodies, notably because of their very high expression rates in Escherichia coli, their high stability paired with high affinity, and successful reports of their use in co-crystallization (15–19). Their architecture results in a very rigid structure that facilitates multiple crystal contacts and may promote crystal formation of the protein of interest by providing additional surfaces for such crystal contacts.We report here the selection and analysis of DARPin binders directed against a macromolecular multiprotein ensemble, the TP901-1 baseplate BppU·BppL protein complex. Ribosome display selection, ELISA screening, and surface plasmon resonance (SPR) measurements allowed us to isolate and characterize three N2C DARPins that recognized the RBP (BppL of the BppU·BppL complex) with high specificity and affinity. Further studies showed that the three DARPins bound to a unique area of the RBP at the tip of the head domain. QELS, MALS, UV, and refractometry coupled online with a size exclusion chromatography (SEC) column allowed us to monitor complex formation in solution as well as to estimate DARPin binding stoichiometry. Crystals of one of these selected DARPins in complex with the RBP were obtained, and the x-ray structure was solved at 2.1 Å resolution. This constitutes the first structure of a DARPin complex originating from the N2C library and the highest resolution for a DARPin complex structure reported to date. Finally, phage adsorption inhibition experiments demonstrated that the three N2C DARPins strongly inhibited L. lactis infection by TP901-1. We describe the DARPin·RBP interface and compare it to other DARPin interfaces. We also compare it to the p2 RBP·VHH5 complex, a previously selected llama VHH domain inhibiting p2 phage adsorption (12), to highlight the different binding mode of these two types of binders.