Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.

<p>Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.</p>

Les anticorps catalytiques sont étudiés pour comprendre leur rôle en conditions physiopathologiques. Ils semblent aussi représenter des outils révolutionnaires pour des études à l'interface entre la chimie, la biochimie, la biologie et immunologie. Par conséquent, la connaissance des relations de structure- fonction représente un grand intérêt. Nous avons exploré deux systèmes d'expression pour la production d'un anticorps catalytique modèle présentant une activité bêta-lactamase. Le fragment scFv recombinant a été produit dans le système d'expression procaryote. Les scFv sont souvent décrits comme des protéines difficiles à produire. Une méthode efficace a été développée pour produire de grandes quantités de scFv solubles et correctement repliés. L'anticorps catalytique entier a aussi été produit en exploitant le système d'expression eucaryote. Des cellules de mammifères ont été utilisées car elles peuvent conserver le repliement original des protéines, leur assemblage et les modifications post-traductionnelles. La structure secondaire du scFv catalytique a été analysée par dichroïsme circulaire pour s’assurer que la renaturation du scFv est en accord avec le repliement des scFv natifs. La fonctionnalité du scFv catalytique et de l'anticorps catalytique entier a été validée par deux approches : (1) le développement d’un test immuno-enzymatique (ELISA) et la résonance plasmonique de surface (RPS) et (2) le développement d'un test catalytique sensible utilisant un substrat fluorogénique. Ce travail amène à considérer de potentielles applications biotechnologiques et thérapeutiques des anticorps catalytiques. / Catalytic antibodies are investigated in order to understand their role under physio-pathological situations. But they also appear to be revolutionary tools to perform studies at the interface between chemistry, biochemistry, biology and immunology. Consequently, the knowledge of structure–function relationships is of great interest. We explored two expression systems for the production of a model catalytic antibody displaying a beta-lactamase activity. The recombinant scFv fragment was produced in the prokaryotic expression system. scFv fragments are often described as proteins being laborious to produce. An efficient method was developed to produce large quantities of refolded soluble catalytic scFv. Whole catalytic antibody was also produced by exploiting eukaryotic expression system. Mammalian cells were used because they are able to retain the original protein folding, assembly and post-translational modifications. The secondary structure of the catalytic scFv has been analyzed by circular dichroism to ensure that the refolded scFv is consistent with a native scFv fold. The functionality of the catalytic scFv and whole catalytic antibody has been validated by two approaches: (1) development of enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (2) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate. This will lead to consider potential biotechnological and therapeutic applications of catalytic antibodies.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Richard 'Dick' Hesse / Carol Wyatt / Over the last two decades molecular laboratory techniques have enabled researchers to investigate the infection, replication and pathogenesis of viral disease. In the early eighties, Dr. George Smith developed a unique system of molecular selection. He showed that the fd bacteriophage genome could be manipulated to carry a sequence of DNA coding for a protein not contained in the phage genome. Infection of the recombinant bacteriophage or phagemid into a specific strain of the bacterium, Escherichia coli, produced progeny phage with the coded protein displayed as a fusion with the phage's coat protein. Antibody phage display utilizes the same technology with the DNA encoding an antibody fragment. The DNA insert can carry the information to produce either a single chain variable fragment (scFv) producing the heavy chain variable and light chain variable (VH-VL) portion or a Fab fragment which also contains the heavy chain constant 1 with the light chain constant (CH and CL) portion of an antibody. Screening an antibody phage display library has the possibility of producing an antibody not produced in the normal course of immune selection. This decade also saw the emergence of a viral disease affecting the porcine population. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has been one of the most costly diseases affecting the pig producer. Molecular investigations found that PRRSV is a single, positive-stranded RNA virus which codes for five structural and 12-13 nonstructural proteins producing an enveloped, icosahedral virus. An interesting characteristic of PRRSV is the ability to produce infective progeny with genomic deletions, insertions and mutations within the nonstructural protein 2 (nsp2). With this knowledge, many researchers have produced marker vaccines containing fluorescent tags with the hope of developing a DIVA (Differentiate Infected from Vaccinated Animals) vaccine. In my Master‟s studies, I studied the techniques of antibody phage display technology and how to apply these methods to producing scFvs which recognize a recombinant PRRSV nsp2 fragment protein and the native protein during infection of MARC-145 cells.