Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened fromphage display libraries is their stable expression on a phageduring multiple selection rounds. Thus, if stringent panningprocedures are employed, selection is simultaneously drivenby antigen affinity, stability and solubility. To take advantageof robust pre-selected scaffolds of such molecules, we graftedsingle-chain Fv (scFv) antibodies, previously isolated froma human phage display library after multiple rounds of in vitropanning on tumor cells, with the specificity of the clinicallyestablished murine monoclonal anti-CD22 antibody RFB4. We showthat a panel of grafted scFvs retained the specificity of themurine monoclonal antibody, bound to the target antigen withhigh affinity (6.4–9.6 nM), and exhibited exceptionalbiophysical stability with retention of 89–93% of theinitial binding activity after 6 days of incubation in humanserum at 37°C. Selection of stable human scaffolds withhigh sequence identity to both the human germline and the rodentframeworks required only a small number of murine residues tobe retained within the human frameworks in order to maintainthe structural integrity of the antigen binding site. We expectthis approach may be applicable for the rapid generation ofhighly stable humanized antibodies with low immunogenic potential. Received June 10, 2003; accepted August 27, 2003.     

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.     

A variant human IgG1-Fc mediates improved ADCC

Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.     

2-Aminobenzothiazoles Inhibit Virulence Gene Expression and Block Polymyxin Resistance in Salmonella enterica

One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg²⁺, a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella’s resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella.     

Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16

The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α₂β₂ and its monomeric subunits CK2α and CK2β. A series of analogues of W16 ((3aR,4S,10S,10aS)-4-{[(S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]carbonyl}-10-(3,4,5-trimethoxyphenyl)-4,5,10,10a-tetrahydrofuro[3,4-b]carbazole-1,3(3aH)-dione ((+)- 3 a )) was prepared in an one-pot, three-component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide ( 9 ) and N-methylimide ( 10 ) substructure. The enantiomer (−)- 3 a (K_i=4.9 μM) of the lead compound (+)- 3 a (K_i=31 μM) showed a more than sixfold increased inhibition of the CK2α/CK2β interaction (protein-protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (−)- 3 a did not show an increased enzyme inhibition of the CK2α₂β₂ holoenzyme, the CK2α subunit or the mutated CK2α′ ^C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (−)- 9 a (K_i=3.6 μM) and the N-methylimide (+)- 10 a (K_i=2.8 μM) represent the most promising inhibitors of the CK2α/CK2β interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)- 12 , with a carboxy moiety in the 4-position, displays the highest CK2α/CK2β interaction inhibition (K_i=1.8 μM) of this series of compounds.     

Current Understanding of Structure–Processing–Property Relationships in BaTiO3–Bi(M)O3 Dielectrics

As part of a continued push for high permittivity dielectrics suitable for use at elevated operating temperatures and/or large electric fields, modifications of BaTiO₃ with Bi(M)O₃, where M represents a net‐trivalent B‐site occupied by one or more species, have received a great deal of recent attention. Materials in this composition family exhibit weakly coupled relaxor behavior that is not only remarkably stable at high temperatures and under large electric fields, but is also quite similar across various identities of M. Moderate levels of Bi content (as much as 50 mol%) appear to be crucial to the stability of the dielectric response. In addition, the presence of significant Bi reduces the processing temperatures required for densification and increases the required oxygen content in processing atmospheres relative to traditional X7R‐type BaTiO₃‐based dielectrics. Although detailed understanding of the structure–processing–property relationships in this class of materials is still in its infancy, this article reviews the current state of understanding of the mechanisms underlying the high and stable values of both relative permittivity and resistivity that are characteristic of BaTiO₃‐Bi(M)O₃ dielectrics as well as the processing challenges and opportunities associated with these materials.     

Tape casting of polysiloxane-derived ceramic with controlled porosity and surface properties

Tape casting has been applied to produce porous hybrid and SiOC ceramic tapes using ceramic precursors and commercially available polysiloxanes as polymeric binders. SiC particles of two different mean sizes (4.5 or 6.5?μm) were used as inert fillers to prevent shrinkage and increase mechanical stability. Macroporosity was adjusted by varying the azodicarbonamide (ADA) content from 0 to 30?wt.%. Decomposition of the polysiloxanes at 600?°C resulted in the generation of micropores with high specific surface area (187–267 m²?g^?1) and a predominant hydrophobic behavior. At 1000?°C mainly meso/macroporosity were observed (SSA: 32–162 m²?g^?1) accompanied by increased hydrophilicity. The influence of ADA content, SiC size, and pyrolysis temperature on open porosity (2.5–37%), average pore size (<0.01–1.76?μm), surface characteristics, and flexural strength (10.5–121?MPa) were investigated. The porous tapes with different surface characteristics and controlled structure are highly promising for applications involving membrane processes, particularly microfiltration systems (0.1–10?μm).     

Design of tailored biodegradable implants: The effect of voltage on electrodeposited calcium phosphate coatings on pure magnesium

Magnesium, as a biodegradable metal, offers great potential for use as a temporary implant material, which dissolves in the course of bone tissue healing. It can sufficiently support the bone and promote the bone healing process. However, the corrosion resistance of magnesium implants must be enhanced before its application in clinical practice. A promising approach of enhancing the corrosion resistance is deposition of bioactive coating, which can reduce the corrosion rate of the implants and promote bone healing. Therefore, a well-designed substrate-coating system allowing a good control of the degradation behavior is highly desirable for tailored implants for specific groups of patients with particular needs. In this contribution, the influence of coating formation conditions on the characteristics of potentiostatically electrodeposited CaP coatings on magnesium substrate was evaluated. Results showed that potential variation led to formation of coatings with the same chemical composition, but very different morphologies. Parameters that mostly influence the coating performance, such as the thickness, uniformity, deposits size, and orientation, varied from produced coating to coating. These characteristics of CaP coatings on magnesium were controlled by coating formation potential, and it was demonstrated that the electrodeposition could be a promising coating technique for production of tailored magnesium-CaP implants.