Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaig

Responsive glyco-poly(2-oxazoline)s: synthesis, cloud point tuning, and lectin binding

A new sugar-substituted 2-oxazoline monomer was prepared using the copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Its copolymerization with 2-ethyl-2-oxazoline as well as 2-(dec-9-enyl)-2-oxazoline, yielding well-defined copolymers with the possibility to tune the properties by thiol-ene "click" reactions, is described. Extensive solubility studies on the corresponding glycocopolymers demonstrated that the lower critical solution temperature behavior and pH-responsiveness of these copolymers can be adjusted in water and phosphate-buffered saline (PBS) depending on the choice of the thiol. By conjugation of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose and subsequent deprotection of the sugar moieties, the hydrophilicity of the copolymer could be increased significantly, allowing a cloud-point tuning in the physiological range. Furthermore, the binding capability of the glycosylated copoly(2-oxazoline) to concanavalin A was investigated

The Density of States in High-Tc Superconductors Vortices

We calculated the electronic structure of a vortex in a pseudogapped superconductor within a model featuring strong correlations. With increasing strength of the correlations, the BCS core states are suppressed and the spectra in and outside the core become similar. If the correlations are short-range, we find new core states in agreement with the observations in YBaCuO and BiSrCaCuO. Our results point to a common phenomenology for these two systems and indicate that normal-state correlations survive below Tc without taking part in the overall phase coherence.Comment: REVTeX 4, 5 pages, 2 EPS figures. Some changes to the text; new figures; references update

Metabolic scaling in modular animals

Metabolic scaling is the relationship between organismal metabolic rate and body mass. Understanding the patterns and causes of metabolic scaling provides a powerful foundation for predicting biological processes at the level of individuals, populations, communities, and ecosystems. Despite intense interest in, and debate on, the mechanistic basis of metabolic scaling, relatively little attention has been paid to metabolic scaling in clonal animals with modular construction, such as colonial cnidarians, bryozoans, and colonial ascidians. Unlike unitary animals, modular animals are structural individuals subdivided into repeated morphological units, or modules, each able to acquire, process, and share resources. A modular design allows flexibility in organism size and shape with consequences for metabolic scaling. Furthermore, with careful consideration of the biology of modular animals, the size and shape of individual colonies can be experimentally manipulated to test competing theories pertaining to metabolic scaling. Here, we review metabolic scaling in modular animals and find that a wide range of scaling exponents, rather than a single value, has been reported for a variety of modular animals. We identify factors influencing variation in intraspecific scaling in this group that relate to the general observation that not all modules within a colony are identical. We highlight current gaps in our understanding of metabolic scaling in modular animals, and suggest future research directions, such as manipulating metabolic states and comparisons among species that differ in extent of module integration

Determination of the Coherence Length and the Cooper-Pair Size in Unconventional Superconductors by Tunnelling Spectroscopy

The main purpose of the paper is to discuss a possibility of the determination of the values of the coherence length and the Cooper-pair size in unconventional superconductors by using tunnelling spectroscopy. In the mixed state of type-II superconductors, an applied magnetic field penetrates the superconductor in the form of vortices which form a regular lattice. In unconventional superconductors, the inner structure of a vortex core has a complex structure which is determined by the order parameter of the superconducting state and by the pairing wavefunction of the Cooper pairs. In clean superconductors, the spatial variations of the order parameter and the pairing wavefunction occur over the distances of the order of the coherence length and the Cooper-pair size, respectively. Therefore, by performing tunnelling spectroscopy along a line passing through a vortex core, one is able, in principle, to estimate the values of the coherent length and the Cooper-pair size.Comment: 13 pages, including 17 figure

High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting.

The tumour-associated antigen epithelial glycoprotein-2 (EGP-2) is a promising target for detection and treatment of a variety of human carcinomas. Antibodies to this antigen have been successfully used in patients for imaging of small-cell lung cancer and for adjuvant treatment of minimal residual disease of colon cancer. We describe here the isolation and complete characterization of high-affinity single-chain variable fragments (scFv) to the EGP-2 antigen. First, the binding kinetics of four murine whole antibodies directed to EGP-2 (17-1A, 323/A3, MOC-31 and MOC-161) were determined using surface plasmon resonance (SPR). The MOC-31 antibody has the lowest apparent off-rate, followed by MOC-161 and 323/A3. The V-genes of the two MOC hybridomas were cloned as scFv in a phage display vector and antigen-binding phage were selected by panning on recombinant antigen. The scFvs compete with the original hybridoma antibodies for binding to antigen and specifically bind to human carcinomas in immunohistochemistry. MOC-31 scFv has an off-rate which is better than those of the bivalent 17-1A and 323/A3 whole antibodies, providing it with an essential characteristic for tumour retention in vivo. The availability of these high-affinity anti-EGP-2 antibody fragments and of their encoding V-genes creates a variety of possibilities for their future use as tumour-targeting vehicles

Bicistronic DNA display for in vitro selection of Fab fragments

In vitro display methods are superior tools for obtaining monoclonal antibodies. Although totally in vitro display methods, such as ribosome display and mRNA display, have the advantages of larger library sizes and quicker selection procedures compared with phage display, their applications have been limited to single-chain Fvs due to the requirement for linking of the mRNA and the nascent protein on the ribosome. Here we describe a different type of totally in vitro method, DNA display, that is applicable to heterodimeric Fab fragments: in vitro compartmentalization in water-in-oil emulsions allows the linking of an oligomeric protein and its encoding DNA with multiple ORFs. Since previously used emulsions impaired the synthesis of functional Fab fragments, we modified conditions for preparing emulsions, and identified conditions under which it was possible to enrich Fab fragments 106-fold per three rounds of affinity selection. Furthermore, we confirmed that genes encoding stable Fab fragments could be selected from a Fab fragment library with a randomized hydrophobic core in the constant region by applying heat treatment as a selection pressure. Since this method has all advantages of both phage display and totally in vitro display, it represents a new option for many applications using display methods

Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms

Anyons in a weakly interacting system

We describe a theoretical proposal for a system whose excitations are anyons with the exchange phase pi/4 and charge -e/2, but, remarkably, can be built by filling a set of single-particle states of essentially noninteracting electrons. The system consists of an artificially structured type-II superconducting film adjacent to a 2D electron gas in the integer quantum Hall regime with unit filling fraction. The proposal rests on the observation that a vacancy in an otherwise periodic vortex lattice in the superconductor creates a bound state in the 2DEG with total charge -e/2. A composite of this fractionally charged hole and the missing flux due to the vacancy behaves as an anyon. The proposed setup allows for manipulation of these anyons and could prove useful in various schemes for fault-tolerant topological quantum computation.Comment: 7 pages with 3 figures. For related work and info visit http://www.physics.ubc.ca/~fran

Magnetic field induced charge and spin instabilities in cuprate superconductors

A d-wave superconductor, subject to strong phase fluctuations, is known to suffer an antiferromagnetic instability closely related to the chiral symmetry breaking in (2+1)-dimensional quantum electrodynamics (QED3). On the basis of this idea we formulate a "QED3 in a box" theory of local instabilities of a d-wave superconductor in the vicinity of a single pinned vortex undergoing quantum fluctuations around its equilibrium position. As a generic outcome we find an incommensurate 2D spin density wave forming in the neighborhood of a vortex with a concomitant "checkerboard" pattern in the local electronic density of states, in agreement with recent neutron scattering and tunneling spectroscopy measurements.Comment: 4 pages REVTeX + 2 PostScript figures included in text. Version to appear in PRL (minor stylistic changes, references updated). For related work and info visit http://www.physics.ubc.ca/~fran