Evolution of Bioaffinity Reagents by Phage Display

Antibodies are natural binding proteins produced in vertebrates as a response to invading pathogens and foreign substances. Because of their capability for tight and specific binding, antibodies have found use as binding reagents in research and diagnostics. Properties of cloned recombinant antibodies can be further improved by means of in vitro evolution, combining mutagenesis with subsequent phage display selection. It is also possible to isolate entirely new antibodies from vast naïve or synthetic antibody libraries by phage display. In this study, library techniques and phage display selection were applied in order to optimise binding scaffolds and antigen recognition of antibodies, and to evolve new and improved bioaffinity reagents. Antibody libraries were generated by random and targeted mutagenesis. Expression and stability were mainly optimised by the random methods whereas targeted randomisation of the binding site residues was used for optimising the binding properties. Trinucleotide mutagenesis allowed design of defined randomisation patterns for a synthetic antibody library. Improved clones were selected by phage display. Capture by a specific anti- DHPS antibody was exploited in the selection of improved phage display of DHPS. Efficient selection for stability was established by combining phage display selection with denaturation under reducing conditions. Broad-specific binding of a generic anti-sulfonamide antibody was improved by selection with one of the weakest binding sulfonamides. In addition, p9 based phage display was studied in affinity selection from the synthetic library. A TIM barrel protein DHPS was engineered for efficient phage display by combining cysteinereplacement with random mutagenesis. The resulting clone allows use of phage display in further engineering of DHPS and possibly use as an alternative-binding scaffold. An anti-TSH scFv fragment, cloned from a monoclonal antibody, was engineered for improved stability to better suite an immunoassay. The improved scFv tolerates 8 – 9 °C higher temperature than the parental scFv and should have sufficient stability to be used in an immunoanalyser with incubation at 36 °C. The anti-TSH scFv fragment was compared with the corresponding Fab fragment and the parental monoclonal antibody as a capturing reagent in a rapid 5-min immunoassay for TSH. The scFv fragment provided some benefits over the conventionally used Mab in anayte-binding capacity and assay kinetics. However, the recombinant Fab fragment, which had similar kinetics to the scFv, provided a more sensitive and reliable assay than the scFv. Another cloned scFv fragment was engineered in order to improve broad-specific recognition of sulfonamides. The improved antibody detects different sulfonamides at concentrations below the maximum residue limit (100 μg/kg in EU and USA) and allows simultaneous screening of different sulfonamide drug residues. Finally, a synthetic antibody library was constructed and new antibodies were generated and affinity matured entirely in vitro. These results illuminate the possibilities of phage display and antibody engineering for generation and optimisation of binding reagents in vitro and indicate the potential of recombinant antibodies as affinity reagents in immunoassays.

Controlling Access to Personal Information

Tässä diplomityössä käsitellään henkilökohtaisen tiedon saannin kontrollointia ja tiedon kuvaamista. Työn käytännön osuudessa suunniteltiin XML –malli henkilökohtaisen tiedon kuvaamiseen. Henkilökohtaisten tietojen käyttäminen mahdollistaa henkilökohtaisen palvelun tarjoamisen ja myös palvelun automatisoinnin käyttäjälle. Henkilökohtaisen tiedon kuvaaminen on hyvin oleellista, jotta palvelut voivat kysellä ja ymmärtää tietoja. Henkilökohtaiseen tietoon vaikuttaa erilaisia tekijöitä, jotka on myös otettava huomioon tietoa kuvattaessa. Henkilökohtaisen tiedon leviäminen eri palveluiden tarjoajille tuo mukanaan myös riskejä. Henkilökohtaisen tiedon joutuminen väärän henkilön käsiin saattaa aiheuttaa vakaviakin ongelmia tiedon omistajalle. Henkilökohtaisen tiedon turvallisen ja luotettavan käytettävyyden kannalta onkin hyvin oleellista, että käyttäjällä on mahdollisuus kontrolloida kenelle hän haluaa luovuttaa mitäkin tietoa.

Targeted Therapy Possibilities for Metastatic Melanoma

The incidence of malignant melanoma of the skin has been steadily rising worldwide during the past decades. Most early detected primary tumors can be removed surgically and the prognosis is good. However, at the same time there still is no permanent cure for metastatic melanoma and its prognosis is poor, although lately new effective drugs have emerged. In this thesis, four different approaches of experimental therapy for metastatic melanoma were studied. Endogenous cis-Urocanic acid (UCA) is found in every individual’s skin, where exposure to UV light from the sun generates it from its inactive trans conformation. Cis- UCA was found to destroy malignant melanoma cells in culture under an acidified pH and sufficient concentration through caspase-3 mediated apoptosis. Furthermore, cis-UCA is able to considerably diminish the growth rate in human melanoma tumors on living SCID mice. Using replication-competent Semliki Forest viruses, human melanoma tumors grown in SCID mice were dramatically shrunken as the fulminant production of viruses in melanoma cells leads them to apoptosis within 72 hours. Small oligopeptides attaching to melanoma cells were identified using in vivo phage display. The melanoma-specific peptides found were further tested in vitro on adenoviruses. Ultimately, the adenoviral retargeting using the peptides was tested in vivo. One peptide homed to human transferring receptor upregulated on melanoma cells. In order to kill the malignant melanoma cells with the retargeted adenoviruses, the viruses should carry genetic material producing apoptotic proteins in the cancer tissue. TIMP-3 has been identified as a good candidate for such a protein, as it inhibits malignant cell adhesion as well as promotes apoptosis through a caspase-8 pathway. It is further shown here that adenovirally delivered TIMP-3 is even more potent, as it could kill non-adherent cancer cells, lacking the fully functional death receptor signalling pathway. Adenovirally delivered TIMP-2 also showed marked antitumor effects in human malignant melanoma xenografts on SCID mice both in ex vivo and systemic delivery.

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Die Beendigung von finnischen und deutschen Telefonaten. Eine interaktionslinguistische, kontrastierende Untersuchung

Suomalaisten ja saksalaisten arkikeskustelujen välillä on sekä yhtäläisyyksiä että eroja. Tässä saksalaisen filologian alaan kuuluvassa tutkimuksessa tarkastellaan yhtä keskeistä arkikeskustelun toimintoa, puhelinkeskustelun lopetusta, suomen- ja saksanpuhujien tuottamana. Aineistona on käytetty suomen- ja saksankielisten äidinkielisten puhujien tätä tutkimusta varten nauhoittamia henkilökohtaisia luonnollisia puhelinkeskusteluja. Aineistoon valikoitui 12 suomalaista ja 12 saksalaista puhelua. Nauhoitteiden käyttöön on saatu asianmukainen lupa kaikilta osapuolilta. Puhelut on litteroitu saksalaisella kielialueella vakiintuneen GAT-litterointisysteemin mukaan. Teoreettis-metodisena kehyksenä on kaksi tutkimusalaa, vuorovaikutuslingvistiikka ja kielten vertailu. Vuorovaikutuslingvistinen tarkastelu keskittyy havaintoihin vuorojen ja puheen sekvenssien rakenteesta. Vuorojen merkitysten tulkinnassa hyödynnetään systemaattisesti prosodian antamia vihjeitä. Tuloksena on yksittäisten lopetusten keskustelunanalyyttinen lähikuvaus, jonka pohjalta määritellään kulloisenkin lopetuksen sekvenssirakenne. Kaikki lopetukset olivat siltä osin yhteneväisiä, että niissä kaikissa havaittiin ainakin aloittava, tulevaan tapaamiseen viittaava sekä lopputervehdyksiin johtava sekvenssi. Sekvenssirakenteen variaatioiden pohjalta aineiston lopetukset voidaan kuitenkin jaotella ryhmiin. Sekä suomen- että saksankielisessä aineistossa havaittiin kolmentyyppisiä lopetuksia: kompakteja, komplekseja ja keskeytettyjä lopetuksia. Ryhmittely kolmeen tyyppiin on avuksi seuraavassa kuvausvaiheessa, jossa verrataan suomen- ja saksankielisiä lopetuksia toisiinsa. Samanaikaisesti kun tutkimus valottaa kohtia, joissa kaksi aineistosettiä yhtenevät ja eroavat, se myös esittää, mitkä vuorovaikutuksen tasot soveltuvat kieltenvälisen vertailun kohteiksi. Pohdintaa siitä, mitä vuorovaikutuksen tasoja kieltenväliseen vertailuun voidaan sisällyttää, onkin toistaiseksi esitetty verrattain vähän. Työ siis rakentaa siltaa vuorovaikutuslingvistisen ja kontrastiivisen kielitieteen välille.

From genotype to phenotype : diversity and population structure of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD) causing high fish mortalities and significant economic losses to the freshwater salmonid aquaculture industry around the world. Today BCWD outbreaks are mainly treated with environmentally hazardous antimicrobial agents and alternative preventative measures are urgently needed in order to ensure the well-being of animals and the sustainability of aquaculture. The diversity of pathogenic bacteria challenges the development of universal control strategies and in many cases the pathogen population structure, i.e. the total genetic diversity of the species must be taken into account. This work integrates the tools of modern molecular biology and conventional phenotypic microbiology to gain knowledge about the diversity and population structure of F. psychrophilum. The present work includes genetic characterization of a large collection of isolates collected from diverse origins and years, from aquaculture in a whole region including different countries, and provides the first international validation of a universal multilocus sequence typing (MLST) approach for unambiguous genetic typing of F. psychrophilum. Population structure analyses showed that the global F. psychrophilum population is subdivided into pathogenic species-specific clones, of which one particular genetic lineage, clonal complex CC-ST2, has been responsible for the majority of BCWD outbreaks in rainbow trout (Oncorhynchus mykiss) in European aquaculture facilities over several decades. Genotypic and phenotypic population heterogeneity affecting antimicrobial resistance in F. psychrophilum within BCWD outbreaks was discovered. Specific genotypes were associated with severe infections in farmed rainbow trout and Atlantic salmon (Salmo salar), and in addition to high adherence, antimicrobial resistance was strongly associated with outbreak strains. The study brought additional support for the hypothesis of an epidemic F. psychrophilum population structure, where recombination is an important force for the generation and maintenance of genetic diversity, and a significant contribution towards mapping the genetic diversity of this important fish pathogen. Evidence indicating dissemination of virulent strains with commercial movement of fish and fish products was strengthened.

Detection, Identification and Molecular Variation of Human Enteroviruses

Picornaviruses are the most common human viruses and the identification of the picornaviruses is nowadays based on molecular techniques, for example, reverse transcriptase polymerase chain reaction (RT-PCR). One aim of this thesis was to improve the identification of picornaviruses, especially rhino- and enteroviruses, with a real-time assay format and, also, to improve the differentiation of the viruses with genus-specific locked nucleic acid (LNA) probes. Another aim was to identify and study the causative agent of the enterovirus epidemics that appeared in Finland during seasons 2008-2010. In this thesis, the first version of picornavirus qRT-PCR with a melting curve analysis was used in a study of rhinovirus transmission within families with a rhinovirus positive index child where rhinovirus infection was monitored in all family members. In conclusion, rhinoviruses spread effectively within families causing mostly symptomatic infections in children and asymptomatic infections in adults. To improve the differentiation between rhino- and enterovirus the picornavirus qRT-PCR was modified with LNA-incorporated probes. The LNA probes were validated with picornavirus prototypes and different clinical specimen types. The LNA probe-based picornavirus qRT-PCR was able to differentiate all rhino- and enteroviruses correctly, which makes it suitable for diagnostic use. Moreover, in this thesis enterovirus outbreaks were studied with a well-observed method to create a strain-specific qRT-PCR from the typing region VP1 protein. In a hand-foot-and-mouth-disease (HFMD) outbreak in 2008, the causative agent was identified as CV-A6 and when the molecular evolution of the new HFMD CV-A6 strain was studied it was found that CV-A6 was the emerging agent for HFMD and onychomadesis. Furthermore, unusual E-30 meningitis epidemics that apeared during seasons 2009 and 2010 were studied with strain-specific qRT-PCR. The E-30 affected mostly adolescents and was probably spread in sports teams.