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

Apšvietimas kaip architektūros modernumo simbolis

The paper is designed to reveal the aesthetics of artificial lighting and its influence on the architecture of the 20th century. The main topics discussed are electric lighting, which appeard in our history at the end of the 19th century, and the technical development of lighting till the middle of the 20th century. Connections of artificial lighting with visual arts, its influence on advertisement, building architecture and the whole city are analysed. An idea is proposed that although lighting by nature was purely functional, very soon it acquired symbolic ambitions to represent architecture. In modern times architects understood that lighting was both a technological development and a symbol of a new era, when there appeared an independent field of creation - lighting architecture. Santrauka Straipsnyje nagrinėjama dirbtinio apšvietimo estetika ir jos įtaka XX a. architektūrai. Išryškinta XIX a. pabaigoje atsiradusio elektrinio apšvietimo svarba ir atskleista apšvietimo techninė raida iki XX a. vidurio. Nagrinėjamos dirbtinio apšvietimo sąsajos su vizualiaisiais menais, apšvietimo įtaka reklamai, pastatų architektūrai bei visam miestui. Straipsnyje keliama idėja, kad nors apšvietimo prigimtis pradžioje buvo grynai funkcionali, ji greitai įgavo simbolinių ambicijų – reprezentuoti architektūrą. Architektai į apšvietimą žvelgė ne tik kaip į technologinę pažangą, tai buvo naują erą ženklinantis simbolis, erą, kurioje atsiranda didelės įtakos visoms kūrybinėms idėjoms turinti nauja savarankiška kūrybos sritis – šviesos architektūra. First Published Online: 22 May 2013 Reikšminiai žodžiai: meninis, dirbtinis, elektrinis apšvietimas, stiklo, šviesos architektūra, modernumas

Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems

Measurement of antibody-membrane interactions by surface plasmon resonance

Due to problems of immobilizing functional tumor antigens in their natural conformation on surfaces for immunoassays, it is often difficult to evaluate the binding of antibodies derived from phage display libraries depleted and selected by panning on cell lines and living tumor cells. Performing cell membrane based ELISA methods does not reveal any up front kinetic binding information and depends on the performance of secondary antibodies and substrates. To overcome these limitations, we developed a new method to visualize direct antibody-cell membrane interactions by surface plasmon resonance using the Biacore 3000 and on-line signal subtraction on antigen-negative cell membrane vesicles. Conditions for the coating of cell membrane preparations to a carboxymethyl dextran hydrogel surface of a commercially available chip and the proof of concept for this application by the analysis of different formats of anti-CD30 and anti-carcinoembryonic antigen (CEA) antibodies interacting with coated membrane vesicles of CD30-positive/CEA-negative and CD30-negative and CEA-positive cell lines are described