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

Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies/VHH Domains

SummaryThe epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8 bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to the flatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy

Tailoring the flow of soft glasses by soft additives

We examine the vitrification and melting of asymmetric star polymers mixtures by combining rheological measurements with mode coupling theory. We identify two types of glassy states, a {\it single} glass, in which the small component is fluid in the glassy matrix of the big one and a {\it double} glass, in which both components are vitrified. Addition of small star polymers leads to melting of {\it both} glasses and the melting curve has a non-monotonic dependence on the star-star size ratio. The phenomenon opens new ways for externally steering the rheological behavior of soft matter systems.Comment: 4 pages, 4 figures, accepted in Phys. Rev. Let

Predicting the development of stress urinary incontinence 3 years after hysterectomy

We aimed to develop a prediction rule to predict the individual risk to develop stress urinary incontinence (SUI) after hysterectomy. Prospective observational study with 3-year follow-up among women who underwent abdominal or vaginal hysterectomy for benign conditions, excluding vaginal prolapse, and who did not report SUI before surgery (n = 183). The presence of SUI was assessed using a validated questionnaire. Significant prognostic factors for de novo SUI were BMI (OR 1.1 per kg/m(2), 95% CI 1.0-1.2), younger age at time of hysterectomy (OR 0.9 per year, 95% CI 0.8-1.0) and vaginal hysterectomy (OR 2.3, 95% CI 1.0-5.2). Using these variables, we developed the following rule to predict the risk of developing SUI: 32 + BMI-age + (7.5 × route of surgery). We defined a prediction rule that can be used to counsel patients about their individual risk on developing SUI following hysterectom

Self-diffusion in binary blends of cyclic and linear polymers

A lattice model is used to estimate the self-diffusivity of entangled cyclic and linear polymers in blends of varying compositions. To interpret simulation results, we suggest a minimal model based on the physical idea that constraints imposed on a cyclic polymer by infiltrating linear chains have to be released, before it can diffuse beyond a radius of gyration. Both, the simulation, and recently reported experimental data on entangled DNA solutions support the simple model over a wide range of blend compositions, concentrations, and molecular weights.Comment: 10 pages, 2 figure

Topological effects in ring polymers: A computer simulation study

Unconcatenated, unknotted polymer rings in the melt are subject to strong interactions with neighboring chains due to the presence of topological constraints. We study this by computer simulation using the bond-fluctuation algorithm for chains with up to N=512 statistical segments at a volume fraction \Phi=0.5 and show that rings in the melt are more compact than gaussian chains. A careful finite size analysis of the average ring size R \propto N^{\nu} yields an exponent \nu \approx 0.39 \pm 0.03 in agreement with a Flory-like argument for the topologica interactions. We show (using the same algorithm) that the dynamics of molten rings is similar to that of linear chains of the same mass, confirming recent experimental findings. The diffusion constant varies effectively as D_{N} \propto N^{-1.22(3) and is slightly higher than that of corresponding linear chains. For the ring sizes considered (up to 256 statistical segments) we find only one characteristic time scale \tau_{ee} \propto N^{2.0(2); this is shown by the collapse of several mean-square displacements and correlation functions onto corresponding master curves. Because of the shrunken state of the chain, this scaling is not compatible with simple Rouse motion. It applies for all sizes of ring studied and no sign of a crossover to any entangled regime is found.Comment: 20 Pages,11 eps figures, Late

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

Polydisperse star polymer solutions

We analyze the effect of polydispersity in the arm number on the effective interactions, structural correlations and the phase behavior of star polymers in a good solvent. The effective interaction potential between two star polymers with different arm numbers is derived using scaling theory. The resulting expression is tested against monomer-resolved molecular dynamics simulations. We find that the theoretical pair potential is in agreement with the simulation data in a much wider polydispersity range than other proposed potentials. We then use this pair potential as an input in a many-body theory to investigate polydispersity effects on the structural correlations and the phase diagram of dense star polymer solutions. In particular we find that a polydispersity of 10%, which is typical in experimental samples, does not significantly alter previous findings for the phase diagram of monodisperse solutions.Comment: 14 pages, 7 figure

Reproducibility of different screening classifications in ultrasonography of the newborn hip

<p>Abstract</p> <p>Background</p> <p>Ultrasonography of the hip has gained wide acceptance as a primary method for diagnosis, screening and treatment monitoring of developmental hip dysplasia in infants. The aim of the study was to examine the degree of concordance of two objective classifications of hip morphology and subjective parameters by three investigators with different levels of experience.</p> <p>Methods</p> <p>In 207 consecutive newborns (101 boys; 106 girls) the following parameters were assessed: bony roof angle (α-angle) and cartilage roof angle (β-angle) according to Graf's basic standard method, "femoral head coverage" (FHC) as described by Terjesen, shape of the bony roof and position of the cartilaginous roof. Both hips were measured twice by each investigator with a 7.5 MHz linear transducer (SONOLINE G60S^®ultrasound system, SIEMENS, Erlangen, Germany).</p> <p>Results</p> <p>Mean kappa-coefficients for the subjective parameters shape of the bony roof (0.97) and position of the cartilaginous roof (1.0) demonstrated high intra-observer reproducibility. Best results were achieved for α-angle, followed by β-angle and finally FHC. With respect to limits of agreement, inter-observer reproducibility was calculated less precisely.</p> <p>Conclusions</p> <p>Higher measurement differences were evaluated more in objective scorings. Those variations were observed by every investigator irrespective of level of experience.</p

Минералогические исследования в пещерной системе Снежная-Меженного-Иллюзия (Западный Кавказ, Бзыбский хребет): предварительные результаты и направления дальнейших работ

В статье приводятся сведения о минеральном составе водных хемогенных и водных механических отложений в пещерной системе Снежная-Меженного-Иллюзия. В состав водных хемогенных отложений входят Mg- и Sr-содержащий кальцит, арагонит, гипс, гидромагнезит, целестин, стронцианит, доломит, гетит, рутил и циркон. Водные механические отложения сложены преимущественно доломитом, кварцем и кальцитом. В схожих по морфологии и микроклимату частях пещерной системы наблюдаются одинаковые вторичные минералы.У статті наводяться відомості про мінеральний склад водних хемогенних і водних механічних відкладень в печерній системі Сніжна-Меженого-Ілюзія. До складу водних хемогенних відкладень входять кальцит, який містить Mg і Sr, арагоніт, гіпс, гідромагнезіт, целестин, стронціаніт, доломіт, гетит, рутил і циркон. Водні механічні відкладення складені переважно доломітом, кварцом і кальцитом. У схожих за морфологєю та мікрокліматом частинах печерної системи спостерігаються однакові вторинні мінерали.The article presents the preliminary characteristic of the mineral composition of chemogenic formations and clastic deposits of Snezhnaya-Mezhennogo-Illusia cave system. Chemogenic formations are composed by Mg- and Sr-calcite, aragonite, gypsum and hydromagnesite, celestite, strontianite, dolomite, goethite, rutile and zircon. Clastic sediments are composed mainly by dolomite, quartz and calcite. Same secondary minerals are observed in those parts of the cave system that have similar morphology and microclimate