<p>In vitro-test för upptäckt och behandling av tumör eller mikrometastaser har de senaste 30 åren gjort stora framsteg tack vare immunokemi och nya framgångsrika cellodlings- tekniker som bättre reproducerar celltillväxt i tre dimensioner (3D) och det omgivande stromat (multicellulär tumörsfäroidodling). TS1-218 scFv (single chain variable fragment) är en monoklonal antikropp som har affinitet till ett protein tillhörande cytoskelettet (cytokeratin). Av TS1-218 har skapats olika varianter (en dimer TS1-218₂ och en mutant HE1-Q) med syftet att öka affinitet och retentionstid på platsen för dess verkan. I det här projektet försökte vi att testa och jämföra egenskaper hos alla 3 joderade antikropparna genom att inkubera odlade Hela Hep 2 tumörcellssfäroider med dessa antikroppar. Alla tre antikroppsvarianter visade god förmåga att penetrera sfäroider och att binda deras epitop i cytokeratin 8. Försöken visade att det fanns affinitetsskillnader mellan TS1-218 monomer, dimer och mutant vilket visade sig som olika inbindningsförmåga till sfäroiderna.</p>

In vitro-test för upptäckt och behandling av tumör eller mikrometastaser har de senaste 30 åren gjort stora framsteg tack vare immunokemi och nya framgångsrika cellodlings- tekniker som bättre reproducerar celltillväxt i tre dimensioner (3D) och det omgivande stromat (multicellulär tumörsfäroidodling). TS1-218 scFv (single chain variable fragment) är en monoklonal antikropp som har affinitet till ett protein tillhörande cytoskelettet (cytokeratin). Av TS1-218 har skapats olika varianter (en dimer TS1-2182 och en mutant HE1-Q) med syftet att öka affinitet och retentionstid på platsen för dess verkan. I det här projektet försökte vi att testa och jämföra egenskaper hos alla 3 joderade antikropparna genom att inkubera odlade Hela Hep 2 tumörcellssfäroider med dessa antikroppar. Alla tre antikroppsvarianter visade god förmåga att penetrera sfäroider och att binda deras epitop i cytokeratin 8. Försöken visade att det fanns affinitetsskillnader mellan TS1-218 monomer, dimer och mutant vilket visade sig som olika inbindningsförmåga till sfäroiderna.

La glycoprotéine VI (GP VI) des plaquettes sanguines humaines est le récepteur principal du collagène, composé le plus thrombogénique d'une paroi vasculaire lésée. Ainsi, GPVI est souvent considérée comme une cible de premier plan pour développer des tests diagnostiques ou des stratégies thérapeutiques innovantes, efficaces et sûres afin d'améliorer encore la prise en charge des accidents ischémiques. Les anticorps monoclonaux et leurs fragments actifs produits par ingénierie moléculaire constituent aujourd'hui une nouvelle classe de biomolécules en plein essor avec des propriétés bien adaptées à des applications thérapeutiques et diagnostiques. Notre groupe a produit plusieurs anticorps monoclonaux anti-GPVI par immunisation génique de souris. Ces anticorps ont une affinité élevé pour leur cible. Ils de distinguent les uns des autres par leur spécificité épitopique ainsi que par les effets engendrés par leur liaison à GPVI. Parmi ces anticorps, l'un présente un fort potentiel diagnostique parce qu'il reconnait les formes mono- et dimériques de GPVI, mais sa liaison aux plaquettes peut induire une activation ou la perte de GPVI. Un autre anticorps présente un fort potentiel thérapeutique parce que ses fragments actifs monovalents obtenus par papaïnolyse neutralisent l'interaction entre les plaquettes et le collagène, sans activer les plaquette. Cependant, l'origine xénogénique de cet anticorps est responsable d'une forte immunogénicité qui en interdit des applications en médecine humaine. Dans cette étude, nous avons conçus un fragments variable d'anticorps simple chaine (scFv) utile pour quantifier l'expression de la GPVI à la surface des plaquettes sanguines. Ce scFv a été reformaté de façon à lui insérer un motif de reconnaissance de la Protéine L (PpL) qui facilite sa détection et sa purification sans avoir recours à un peptide "drapeau". Nous avons également humanisé et créé plusieurs fragments d'anticorps recombinants monovalents inhibiteurs de l'interaction GPVI / collagène. Ces fragments d'anticorps présentent un potentiel thérapeutique élevé. / Human platelets glycoprotein VI (GPVI) is evidenced to be a platelet receptor of major importance in the occurrence of arterial thrombosis. Thus, it can be considered to be of great interest in diagnosis and therapeutic of atheriosclerotic diseases. Antibodies are powerful molecules which can be used in both diagnostic as well as for therapeutic purposes due to their unique characteristics. Monoclonal and recombinant antibodies have antigen restricted specificity, high affinity and can be used in various assays. Moreover, the good knowledge of their structure and molecular engineering facilities now allows the antibody modulation according to desired properties.Our group has already produced several monoclonal antibodies to human GPVI by gene gun immunization against the immunoadhesin hGPVI-Fc, which differ in fine epitopespecificity, affinity and other functional properties (Lecut et al. 2003). One, 3J24, with diagnostic potential while the other, 9O12, has a therapeutic potential because it blocks the binding of GPVI to collagen. Its Fab fragment has been extensively characterized in vitro,ex vivo and in vivo for its antithrombotic properties.Here, we designed and reshaped a single-chain antibody fragment (scFv) based on 3J24variable domains for the quantification of GPVI with diagnostic potential. We were also involved in the design, production and functional evaluation of humanized anti-GPVI recombinant antibody fragments (scFvs and Fabs) with therapeutic properties.

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>

Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry. / Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.

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.

Human platelets glycoprotein VI (GPVI) is evidenced to be a platelet receptor of major importance in the occurrence of arterial thrombosis. Thus, it can be considered to be of great interest in diagnosis and therapeutic of atheriosclerotic diseases. Antibodies are powerful molecules which can be used in both diagnostic as well as for therapeutic purposes due to their unique characteristics. Monoclonal and recombinant antibodies have antigen restricted specificity, high affinity and can be used in various assays. Moreover, the good knowledge of their structure and molecular engineering facilities now allows the antibody modulation according to desired properties.Our group has already produced several monoclonal antibodies to human GPVI by gene gun immunization against the immunoadhesin hGPVI-Fc, which differ in fine epitopespecificity, affinity and other functional properties (Lecut et al. 2003). One, 3J24, with diagnostic potential while the other, 9O12, has a therapeutic potential because it blocks the binding of GPVI to collagen. Its Fab fragment has been extensively characterized in vitro,ex vivo and in vivo for its antithrombotic properties.Here, we designed and reshaped a single-chain antibody fragment (scFv) based on 3J24variable domains for the quantification of GPVI with diagnostic potential. We were also involved in the design, production and functional evaluation of humanized anti-GPVI recombinant antibody fragments (scFvs and Fabs) with therapeutic properties.