Discovery of targets for tumor immunotherapy using phage display technology

This thesis has focused on the development of methods and their applications for the identification of antibodies reactive with tumor associated antigens (TAA). The phage display technology provides a powerful mean to generate antibodies potentially useful for tumor immunotherapy. A phage antibody displays on its surface an antibody fragment encoded by a gene inserted in the phage genome. The phage antibody can be selected by antigen binding from a diverse population of phage, each displaying antibodies with unique specificities. Any antibody gene repertoire that can be genetically assembled, including naive, immune and synthetic repertoires of human antibody genes can be expressed and displayed on phage. Antibody identification by phage display has primarily been performed with purified antigens for selection. This thesis demonstrates that a crude antigen source, i.e. sections of tissues and intact cells can be used directly and dissected as a complex antigen for phage selection, providing a tool for mutual discovery of new antibodies and their targets. A gradual method development in model systems, from straight positive selection using cells or tissue sections to ultimately achieve the most sophisticated subtractive selection method using tissue sections is described in the enclosed papers. The application of these using a phage antibody library constructed from malignant melanoma or colon cancer immunized Cynomolgus Macaque monkeys led to the identification of putative novel tumor specificities. The identified TAA were highly, homogeneously, frequently and selectively expressed on the cell surface in clinical tumor samples and represent potentially excellent targets for tumor immunotherapy

I ljuset av läxans renässans : faktorer som får elever att ägna sig åt självstudier och göra sina läxor

Självstudier upplevs av många lärare och elever, även i denna studie, som en mycket viktig del av lärandet. Hemmet eller andra avskilda platser som bibliotek kan erbjuda en lugnare studiemiljö än skolans, vilket är essentiellt för koncentrationen. Flera av lärandets nödvändiga delmoment, att nå förståelse, lära in fakta och repetition gynnas av självständigt arbete enligt forskning om läxans fördelar och nackdelar. Denna forskning pekar även på vikten av att gradvis öva upp den mentala och praktiska förmågan till självstudier. Det senare kan anses vara avgörande inför ett självstyrt livslångt lärande i yrkesliv och högre studier såväl som i den privata sfären. I det här arbetet intervjuades fem lärare individuellt och åtta naturvetarelever i grupp om vilka faktorer de ansåg var avgörande för att elever ägnar sig åt självstudier. Lärarna menade att föräldrarnas kravbild och överföring av värderingar var viktigast, medan eleverna hävdade att det var lärarnas kravbild och uppföljning av läxor som var avgörande. Eleverna exemplifierade med att de på grund av skillnaderna mellan två av deras lärares agerande alltid gjorde sina läxor i ämnet latin men sällan i ämnet engelska trotts att detta skapade stor frustration, eftersom de upplevde engelska som ett mycket viktigare ämne. Intervjuresultaten implicerar därför att det främst är det yttre motivationstrycket, från lärare och föräldrar, som får de undersökta naturvetareleverna att ägna sig åt självstudier

Endothelial Cells Expressing an Inflammatory Phenotype Are Lysed by Superantigen-Targeted Cytotoxic T Cells

The objective of this study was to investigate whether the superantigen staphylococcal enterotoxin A (SEA), which binds to HLA class II and T-cell receptor Vβ chains, can direct cytotoxic T cells to lyse cytokine-stimulated endothelial cells (EC). In addition, we wanted to determine whether SEA-primed cytotoxic T cells could be targeted to EC surface molecules as a means of a novel cancer immunotherapy. Human umbilical vein EC (HUVEC), dermal microvascular EC (HMVEC), or the EC line EA.hy926 stimulated with gamma interferon (IFN-γ) or tumor necrosis factor alpha (TNF-α) displayed upregulated HLA class II and adhesion molecule (CD54 and CD106) expression, respectively. SEA-primed T cells induced a strong cytotoxicity against IFN-γ- and TNF-α-activated EA.hy926 which had been preincubated with SEA. Blocking of CD54 completely abrogated the T-cell attack. SEA-D227A, which has a mutated class II binding site, did not promote any cytotoxicity. A strong lysis was observed when a fusion protein consisting of protein A and SEA-D227A was added together with T cells to TNF-α-induced EA.hy926 and HUVEC precoated with monoclonal antibodies (MAb) directed against HLA class I, CD54, or CD106 molecules. Finally, an scFv antibody fragment reactive with an unknown EC antigen was fused with SEA-D227A. Both EA.hy926 and HMVEC were efficiently lysed by scFv-SEA-D227A-triggered cytotoxic T cells. Taken together, superantigen-activated T-cell-dependent EC killing was induced when EC expressed an inflammatory phenotype. Moreover, specific MAb targeting of the superantigen to surface antigens induced EC lysis. Our data suggest that directed T-cell-mediated lysis of unwanted proliferating EC, such as those in the tumor microvasculature, can be clinically useful

A3 -- a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells

The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer