Ng, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 206-211). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of contents --- p.vii / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter / Chapter 1. --- Introduction / Chapter 1.1 --- Growth hormone --- p.1 / Chapter 1.1.1 --- Historic discovery of growth hormone --- p.1 / Chapter 1.1.2 --- Structural and functional study of GH --- p.1 / Chapter 1.1.2.1 --- Molecular evolution of GH --- p.1 / Chapter 1.1.2.2 --- Two-dimensional and three dimensional structures --- p.5 / Chapter 1.1.2.3 --- Heterogeneity of GH --- p.8 / Chapter 1.1.2.4 --- Regulation and secretion pattern of GH --- p.9 / Chapter 1.1.2.5 --- Circulation of GH in blood --- p.11 / Chapter 1.1.2.6 --- Biological activity of GH in human --- p.12 / Chapter 1.2 --- GH receptor and signal transduction --- p.12 / Chapter 1.3 --- GH disorder --- p.15 / Chapter 1.4 --- Treatment for GH disorder --- p.16 / Chapter 1.5 --- GH assay --- p.17 / Chapter 1.6 --- Aims of study --- p.19 / Chapter 2. --- SUMO-hGH expression vector construction / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Fusion partner - SUMO --- p.23 / Chapter 2.3 --- Materials --- p.24 / Chapter 2.3.1 --- Reagents for bacterial culture --- p.24 / Chapter 2.3.2 --- Reagents for agarose gel electrophoresis --- p.26 / Chapter 2.3.3 --- 2'-deoxyribonucleoside 5'-triphosphate mix for polymerase chain reaction --- p.26 / Chapter 2.3.4 --- Sonication buffer --- p.26 / Chapter 2.3.5 --- Modified solubilization buffer --- p.27 / Chapter 2.3.6 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis --- p.27 / Chapter 2.4 --- Methods --- p.29 / Chapter 2.4.1 --- General techniques in molecular cloning of hGH gene --- p.29 / Chapter 2.4.2 --- Expression of SUMO-hGH fusion protein - small scale --- p.42 / Chapter 2.4.3 --- General protein analysis --- p.43 / Chapter 2.5 --- Results --- p.45 / Chapter 2.5.1 --- Molecular cloning of hGH gene into expression vector --- p.45 / Chapter 2.5.2 --- Expression of SUMO-hGH --- p.46 / Chapter 2.5.3 --- Modification of the expression conditions --- p.46 / Chapter 2.6 --- Discussion --- p.50 / Chapter 2.6.1 --- Expression vector --- p.53 / Chapter 2.6.2 --- Protein expression --- p.53 / Chapter 2.7 --- Conclusion --- p.54 / Chapter 3. --- SUMO-hGH purification and downstream processing / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Immobilized-metal affinity chromatography --- p.55 / Chapter 3.3 --- SUMO protease --- p.57 / Chapter 3.4 --- Materials --- p.59 / Chapter 3.4.1 --- Reagents for IMAC purification of SUMO-hGH fusion protein --- p.59 / Chapter 3.4.2 --- Reagents for IMAC purification of mature rhGH --- p.60 / Chapter 3.4.3 --- Reagents for Western blotting --- p.60 / Chapter 3.4.4 --- Gel filtration running buffer --- p.62 / Chapter 3.5 --- Methods --- p.62 / Chapter 3.5.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.62 / Chapter 3.5.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.63 / Chapter 3.5.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.64 / Chapter 3.5.4 --- Purification of rhGH by size exclusion chromatography - gel filtration chromatography --- p.64 / Chapter 3.5.5 --- General protein analysis --- p.65 / Chapter 3.6 --- Results --- p.67 / Chapter 3.6.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.67 / Chapter 3.6.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.69 / Chapter 3.6.3 --- Digestion efficiency of different constructs of SENP1C --- p.73 / Chapter 3.6.4 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.77 / Chapter 3.6.5 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.78 / Chapter 3.7 --- Discussion --- p.81 / Chapter 3.7.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.81 / Chapter 3.7.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.82 / Chapter 3.7.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.82 / Chapter 3.7.4 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.85 / Chapter 3.8 --- Conclusion --- p.85 / Chapter 4. --- Fermentation expression of SUMO-hGH and scale-up of downstream process / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Bioreactor system for E.coli host cultivation --- p.87 / Chapter 4.3 --- Mechanical cell disruption for cell --- p.88 / Chapter 4.4 --- rhGH binding assay --- p.88 / Chapter 4.5 --- Materials --- p.89 / Chapter 4.5.1 --- Reagents for bacterial culture by fermenter --- p.89 / Chapter 4.5.2 --- Reagents for HEK293 Hi cultivation --- p.91 / Chapter 4.5.3 --- Reagents for Dual-Luciferase® Reporter Assay System --- p.92 / Chapter 4.5.4 --- Reagents for silver stain of SDS-PAGE mini-gel --- p.93 / Chapter 4.6 --- Methods --- p.94 / Chapter 4.6.1 --- Bioreactor system and fixed volume fed-batch fermentation --- p.94 / Chapter 4.6.2 --- Large scale mechanically disruption of cell membrane --- p.97 / Chapter 4.6.3 --- Downstream processing of SUMO-hGH --- p.97 / Chapter 4.6.4 --- Culture of HEK293 Hi cells --- p.97 / Chapter 4.6.5 --- Dual-Luciferase® Reporter Assay System --- p.98 / Chapter 4.6.6 --- Silver staining of SDS-PAGE mini-gels --- p.101 / Chapter 4.7 --- Results --- p.101 / Chapter 4.7.1 --- Fed-batch fermentation of E. coli BL21 --- p.101 / Chapter 4.7.2 --- Comparison on disruption methods and the purification of SUMO-hGH from cell lysate --- p.106 / Chapter 4.7.3 --- Optimization of His-MBP-SENP1C digestion condition --- p.108 / Chapter 4.7.4 --- Optimization of rhGH purification in 2nd round of IMAC --- p.110 / Chapter 4.7.5 --- Characterization of mature rhGH --- p.112 / Chapter 4.8 --- Discussion --- p.116 / Chapter 4.8.1 --- Fed-batch fermentation of E. coli BL21 --- p.118 / Chapter 4.8.2 --- Downstream processing of fermentation culture and characterization of rhGH --- p.120 / Chapter 4.8.3 --- M9 based defined medium fermentation study --- p.122 / Chapter 4.8.4 --- rhGH production yield estimation --- p.128 / Chapter 4.8.5 --- Comparison of our fermentation expression system to the published data --- p.130 / Chapter 4.9 --- Conclusion --- p.132 / Chapter 5. --- His-MBP-SENPIC expression and purification / Chapter 5.1 --- Introduction --- p.133 / Chapter 5.2 --- Materials --- p.134 / Chapter 5.2.1 --- Reagents for bacterial culture --- p.134 / Chapter 5.2.2 --- Reagents for immobilized metal affinity chromatography purification of His-MBP-SENP1C --- p.135 / Chapter 5.3 --- Methods --- p.136 / Chapter 5.3.1 --- Expression of His-MBP-SENP1C --- p.136 / Chapter 5.3.2 --- Semi-purification of His-MBP-SENP1C by Ni2+-NTA affinity chromatography --- p.138 / Chapter 5.4 --- Results --- p.139 / Chapter 5.4.1 --- Expression of His-MBP-SENP1C --- p.139 / Chapter 5.4.2 --- Digestion activity of His-MBP-SENP1C expressed --- p.139 / Chapter 5.5 --- Discussion --- p.141 / Chapter 5.5.1 --- Expression and purification of His-MBP-SENP1C --- p.141 / Chapter 5.5.2 --- His-MBP-SENP1C production yield estimation --- p.143 / Chapter 6. --- Production and characterization of monoclonal antibodies against rhGH / Chapter 6.1 --- Introduction --- p.145 / Chapter 6.2 --- Materials --- p.146 / Chapter 6.2.1 --- Reagents for Sp2/0-Ag14 cultivation --- p.146 / Chapter 6.2.2 --- Reagents for PEG fusion --- p.147 / Chapter 6.2.3 --- Reagents for enzyme linked immunosorbent assay --- p.149 / Chapter 6.2.4 --- Reagents for mAbs purification by HiTrap´ёØ Protein G HP Column --- p.150 / Chapter 6.3 --- Methods --- p.151 / Chapter 6.3.1 --- ELISA --- p.151 / Chapter 6.3.2 --- Immunization --- p.152 / Chapter 6.3.3 --- Culturing of myeloma fusion partner cells --- p.153 / Chapter 6.3.4 --- Isolation of splenocyte --- p.153 / Chapter 6.3.5 --- PEG fusion --- p.154 / Chapter 6.3.6 --- Limiting dilution --- p.155 / Chapter 6.3.7 --- Cryopreservation of hybridoma cell lines --- p.156 / Chapter 6.3.8 --- Mass production of monoclonal antibodies --- p.157 / Chapter 6.3.9 --- Purification of IgG mAbs from ascites --- p.157 / Chapter 6.3.10 --- MAbs isotyping --- p.159 / Chapter 6.3.11 --- Determination of kinetic parameters of mAbs --- p.159 / Chapter 6.4 --- Results --- p.162 / Chapter 6.4.1 --- Production of murine anti-rhGH monoclonal antibodies --- p.162 / Chapter 6.4.2 --- Characterization of anti-rhGH mAbs --- p.170 / Chapter 6.5 --- Discussion --- p.178 / Chapter 6.5.1 --- Mass production of mAbs --- p.179 / Chapter 6.5.2 --- Future works on mAbs --- p.179 / Chapter 6.6 --- Conclusion --- p.181 / Chapter 7. --- Development of sandwich ELISA for rhGH / Chapter 7.1 --- Introduction --- p.182 / Chapter 7.2 --- Materials --- p.184 / Chapter 7.2.1 --- Reagents for sandwich ELISA --- p.184 / Chapter 7.3 --- Methods --- p.184 / Chapter 7.3.1 --- Production of rabbit polyclonal antiserum against rhGH --- p.184 / Chapter 7.3.2 --- Sandwich ELISA --- p.185 / Chapter 7.4 --- Results --- p.186 / Chapter 7.4.1 --- Production of rabbit antiserum against rhGH --- p.186 / Chapter 7.4.2 --- Sandwich ELISA --- p.188 / Chapter 7.4.3 --- Optimization of sandwich ELISA --- p.190 / Chapter 7.4.4 --- Specificity of sandwich ELISA --- p.194 / Chapter 7.4.5 --- Cross reactivity of sandwich ELISA to E.coli cell lysate --- p.196 / Chapter 7.4.6 --- Measurement of SUMO-hGH with sandwich ELISA --- p.198 / Chapter 7.5 --- Discussion --- p.201 / Chapter 7.5.1 --- Application of sandwich ELISA --- p.203 / Chapter 7.5.2 --- Future works on sandwich ELISA --- p.205 / Chapter 7.6 --- Conclusion --- p.205 / References --- p.206 / Appendix - pJ2:G01458 nucleotide sequence --- p.213

A prova sorológica de referência na leptospirose ainda é a soroaglutinação microscópica (SAM). Devido à complexidade desta prova avaliamos alguns testes rápidos para triagem dos anticorpos anti-leptospiras na fase aguda da infecção. Na década de 80, uma hemaglutinação passiva, utilizando frações polissacarídicas de leptospiras, foi considerada apropriada ao diagnóstico precoce, porém esta preparação antigênica incluía muitos “antígenos comuns" reconhecidos por anticorpos de 4% dos indivíduos normais. Um novo ELISA (enzyme-linked immunosorbent assay) utilizando uma suspensão de antígenos imunodominantes, resistentes à proteinase K, foi padronizado e avaliado quanto ao seu valor diagnóstico. Com 89,9% de sensibilidade e 97,4% de especificidade, esta técnica, referida como PK-ELISA, satisfaz os requisitos necessários para as provas de triagem da leptospirose humana. No entanto, em virtude de alguns reagentes usados nesta preparação antigênica serem importados e muito instáveis, foi proposta a introdução de novos métodos empregando-se anticorpos monoclonais. Em um “Acordo de Pesquisa Cooperativa" entre o Instituto Adolfo Lutz e o Laboratório Fleury foram produzidos hibridomas contra leptospiras. Dois deles foram selecionados para dar continuidade ao estudo: um, secretando anticorpos monoclonais (AcM) para um epítopo detectado em 16 de 23 sorovares do gênero Leptospira mais freqüentes em nosso meio (clone A12P4), e outro específico a somente um sorogrupo patogênico, icterohaemorragiae (clone H7P1). O AcM A12P4, uma imunoglobulina G2B (IgG2B), reagiu com epítopo presente nos componentes de pesos moleculares (PM) de 16-18 kDa dos lisados de leptospiras das cepas RGA e M-20, quando separados na eletroforese em gel de poliacrilamida, e com componentes de PM de 75-84kDa dos sorovares copenhageni e canicola. Por sua vez, o AcM H7P1, uma imunoglobulina G, reagiu com um epítopo comum a várias frações de PM acima de 21 kDa da cepa RGA e com componentes de PM de 21-22 kDa e de 75-82 kDa da cepa M-20. Os monoclonais foram empregados em provas imunoenzimáticas para a detecção de anticorpos específicos em amostras séricas pareadas coletadas de 52 pacientes com leptospirose, e do grupo controle que incluiu amostras séricas de 57 pacientes com outras doenças consideradas no diagnóstico diferencial, e de 68 indivíduos normais. Estas provas, no entanto, não foram satisfatórias. Finalmente, um novo ELISA foi desenvolvido no presente estudo que utiliza a suspensão de antígenos “AgMc", purificados por cromatografia de afinidade utilizando a Sepharose 4B ativada com CNBr acoplada aos anticorpos monoclonais descritos acima. Os resultados obtidos com esta prova foram comparados aos obtidos com outros testes disponíveis em nosso meio, como a SAM e o ELISA clássico (ELISA c). Este novo método, o “ELISA AgMc", com 80,70 % e 83,33 % de sensibilidade e especifidade, respectivamente, em relação à SAM; valores preditivos positivo e negativo de 69,70% e 90,10% respectivamente e índice de concordância geral de 82,49%, não parece ser um protocolo promissor para o diagnóstico rápido na leptospirose humana. Além disso, tomando-se a SAM como diagnóstico verdadeiro, os resultados obtidos no novo teste, após a conclusão diagnóstica do grupo de pacientes com a leptospirose, mostrou uma discordância significativa. São discutidas as possíveis explicações para os resultados encontrados. / The best serological test for leptospirosis laboratory diagnosis remains the microscopic agglutination test (MAT). Because of the complexity of MAT, we have been developed some rapid screening tests for leptospiral antibodies detection in the acute phase of infection. In the decade of 80, a passive hemagglutination test employing polysaccharide fractions of leptospires was considered appropriate for early diagnosis, but its antigen preparation included “common antigens" recognized by antibodies from 4% of healthy individuals. A new ELISA (enzyme-linked immunosorbent assay) employing proteinase K resistant immunodominant antigens was developed and its potential diagnosis evaluated. This technique, the PK-ELISA, presented 89.9% sensitivity and 97.4% specificity, and satisfied the requeriments needed for serological screening tests of human leptospirosis. However, some of the reagents used in its antigen preparation are imported and very unstable. So, it was proposed, in a “Cooperative Research Accordance" between Instituto Adolfo Lutz and Laboratório Fleury, to try new approaches with monoclonal antibodies. Two hibridomas secreting specific monoclonal antibodies (MAb) were selected: one, against an epitope detected in 16 of 23 members of the genus Leptospira (clone A12P4) and the other, specific to the icterohaemorragiae serogroup (clone H7P1). The MAb A12P4, a G2 (IgG2B) immunoglobulin, reacted with an epitope present in the 16-18 kDa components of icterohaemorragiae serogroup and with the 75-84 kDa components of serovars copenhageni and canicola, after whole-cell lysates of the leptospires were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The MAb H7P1, which is an IgG, reacted with an epitope common to several fractions of molecular weight above 21 kDa of strain RGA and with the 21-22 kDa and the 75-82 kDa components of strain M-20. Both monoclonal antibodies were employed in enzyme immunoassays for detecting specific antibodies in serum samples serially colleted from 52 patients with leptospirosis, and from the control group, which consisted of sera from 57 patients with other diseases included in the differential diagnosis, and from 68 healthy individuals. These tests, however, were not satisfactory. A new ELISA was developed in the present study employing an antigen suspension “AgMc", purified by affinity chromatography with CNBr-activated Sepharose 4B coupled to the monoclonal antibodies described above. The results obtained with this test were compared to the MAT and to the classical IgM ELISA (ELISA c). The new method, “AgMc ELISA", presented serological indices, relatively to reference test MAT, of 80.70 % and 83.33 % of sensitivity and specificity, respectively; positive and negative predictive values of 69.70 % and 90.10 %, respectively, and general agreement index of 82.49 %. So, this test was not considered a promising approach to rapid diagnosis of human leptospirosis. Moreover, the proportion of patients diagnosed as having leptospirosis by the “AgMc ELISA" and the MAT differ significantly. The possible explanations for the results obtained are discussed.

L'industrie biopharmaceutique voit la plupart des thérapies basées sur les anticorps monoclonaux passer du statut de blockbuster à un marché de niche et personnalisé, dans un monde globalisé. Pour poursuivre le développement de nouveaux médicaments, les installations de production existantes et futures doivent accroître leur flexibilité et leur productivité. La chromatographie multi-colonne est l'un des outils potentiels pour y parvenir, comme elle l’a été au cours des dernières décennies pour la pétrochimie et l'industrie alimentaire. En parallèle, l'étape de capture protéine A reste incontournable pour toutes les lignes industrielles de purification grâce à sa spécificité et sa capacité à atteindre facilement un haut niveau de pureté en une seule étape. Ce travail de recherche est une évaluation des procédés de chromatographie multi-colonnes combinés à l'étape de capture protéine A pour augmenter la productivité et l'applicabilité des plateformes de purification actuelles aux activités de production clinique et commerciale. Dans chaque partie du travail, la technologie des colonnes de chromatographie pré-packées a été évalué en tant que facilitateur de procédé multi-colonnes, libérant les équipes opérationnelles des activités de package et des infrastructures associées. Un premier chapitre décrit la traditionnelle revue de la littérature et l'état actuel des connaissances dans le domaine concerné, ainsi qu'une description théorique de la chromatographie en général, et des processus multi-colonnes en particulier. Dans un deuxième chapitre, plusieurs résines protéine A récentes, disponibles sur le marché, ont été comparées dans le cadre de deux procédés multi-colonnes –la chromatographie séquentielle sur plusieurs colonnes (SMCC) et le procédé par Batch Parallèle– et comparées à un procédé traditionnel mono-colonne. Sur la base d'un logiciel de simulation et d'optimisation, les deux processus proposés ont été comparés en termes de gains et de performances. Des recommandations sur la résine et le type de procédé à choisir ont été proposées. Dans un troisième chapitre, l'impact des procédés multi-colonnes sur la qualité et la pureté résultante a été abordé par plusieurs séries d'expériences. L'impact de l'organisation séquentielle d'un processus SMCC a été évalué. L'impact du temps de séjour sur les étapes de lavage et d'élution a également été évalué, afin d'accélérer potentiellement l'étape de capture. Troisièmement, l'impact de la saturation de la résine sur la conception de l'étape de lavage a été évalué. Finalement, des études de cycling ont été réalisées pour détecter si les différents processus multi-colonnes avaient un impact différent sur la durée de vie et les performances de la résine. Dans un quatrième chapitre, un outil de calcul simplifié a été conçu pour proposer un dimensionnement simple des procédés multi-colonnes, tenant mieux compte des contraintes de production de Merck. Cet outil a été utilisé pour évaluer la performance des procédés Batch Parallèle pour deux études de cas. Enfin, dans un chapitre plus exploratoire, l'outil simplifié développé précédemment a été adapté pour évaluer la faisabilité et les contraintes principales des étapes de capture en continu, caractérisées soit par une étape de chargement continu, soit par une étape d'élution continue / The biopharma industry sees most of the therapeutics based on monoclonal antibodies shifting from the blockbuster status to a niche and personalized market, in a globalized world. To continue the development of new drugs, existing and future production facilities have to increase in flexibility and productivity. Multi-column chromatography is one of the potential tools to make that happen, as the technology did in the last decades for the petro and for the food industries. In parallel, the protein A capture step remains a must for all purification trains of the industrial manufacturing capacities due to its specificity and capability to easily reach a high level of purity in a single step. This research work is an evaluation of the multi-column chromatography processes combined with the protein A capture step to increase the productivity and the applicability of current purification platforms for clinical and commercial manufacturing activities. In every part of the work, prepacked chromatography columns have been evaluated as an enabler of multi-column processes, freeing the operational teams from the packing activities burden and associated infrastructures. A first chapter describes the traditional literature review and current state-of-the-art in the relevant field, together with a theoretical description of chromatography in general, and multi-column processes in particular. In a second chapter, several recent, commercially-available protein A resins have been compared when involved in 2 multi-column processes: the Sequential Multi-Column Chromatography (SMCC) process and the Parallel Batch process, and compared to a traditional mono-column process. Based on a simulation and optimization software, both processes proposed have been compared in terms of gains and performance. Recommendations on the resin and the type of process to be selected have been proposed. In a third chapter, the impact of multi-column processes on the resulting quality and purity has been addressed through several sets of experiments. The impact of the sequential organization of an SMCC process has been evaluated. The impact of the residence time on the washing and the elution steps has also been evaluated, in order to potentially speed-up the capture step. Third, the impact of the resin saturation on the design of the washing step has been assessed. Eventually, cycling studies have been performed to detect if the different multi-column processes were impacting differently the resin’s lifetime and performance. In a fourth chapter, a simplified calculation tool has been designed to propose simple sizing of multi-column processes, accounting more accurately for Merck’s production constraints. This tool has been used to evaluate the performance of Parallel Batch processes for 2 case-studies. Eventually, in a more exploratory chapter, the simplified tool previously developed has been adapted to evaluate the feasibility and the primary constraints of continuous capture steps, characterized by either a continuous loading step or a continuous elution step

Multiple research studies have demonstrated racial, socioeconomic status (SES), and neighborhood disparities in first-line treatment of colorectal cancer patients, including those with metastatic colorectal cancer. However, disparities in adjunct monoclonal antibody treatment disparities have not been explored. The purpose of this study was to assess racial, SES, and neighborhood disparities in adjunct monoclonal antibody treatment of elderly metastatic colorectal cancer patients. The research was rooted in 3 theories: the fundamental cause theory, the diffusion of innovations theory, and theory of health disparities and medical technology. Data from the SEER-Medicare database and logistic regression were used to assess the relationship between the variables of interest and adjunct monoclonal antibody therapy. In this study, race (p = 0.070), SES (p = 0.881), and neighborhood characteristics (p = 0.309) did not significantly predict who would receive monoclonal antibody therapy. The results demonstrated a potential improvement in historically documented colorectal cancer treatment disparities. Specifically, historical treatment disparities may not be relevant to newer therapies prescribed to patients with severe disease. The difference could be related to improved access to care or a change in treatment paradigm due to the severity of metastatic colorectal cancer. Future studies aimed at understanding the causes of this social change (i.e., reduced treatment disparities) are warranted. Understanding the root cause of the reduced treatment disparities observed in this study could be used to reduce treatment disparities in other cancer populations.

Mouse monoclonal antibodies directed against Pichinde virus (PCV) were produced to evaluate their application as vehicles for the delivery of antiviral drugs or toxins to virus-infected cells. Four monoclonal antibodies, PC4.9A6, PC4.9D3, PC4.7C2, and PC4.8D3, were of the IgG2a subisotype and reacted with acetone-fixed and live PCV-infected Vero-76 cells. In vivo stained splenic macrophages derived from PCV-infected hamsters that had been injected with fluorescein-labeled PC4.9A6 (FITC9A6) demonstrated a 400% increase in total fluorescence over similarly treated, non-infected cells when analyzed by flow cytometry. This is an indication that FITC-9A6 does have some ability to specifically target PCV infected cells in vivo. Radioimmunoprecipitation of viral proteins showed that all the antibodies precipitated two different PCV proteins, one of 64,000 daltons and another of 38,000 daltons. These proteins are, respectively, PCV ix nucleoprotein (NP) and a breakdown product of NP that is present in PCV infected cells. An immunofluorescent assay (IFA) for PCV was developed. This IFA was used for antiviral drug assays against PCV. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to fixed, virus infected cells at 24 h after infection and counting the fluorescent cells. The 50% effective dose (EDso) for ribavirin against PCV using this IFA was 6.0 IJ. g/ml. The EDso of ribavirin using inhibition of marginal PCV cytopathogenic effect after 12 days was 6.0 IJ. g/ml and using plaque reduction after 5 days is 2.5 IJ. g/ml, indicating that this IFA was of comparable sensitivity. An immunotoxin (IT) was produced by the conjugation of gelonin to PC4.9A6. This IT was tested in vivo in PCV-infected MHA hamsters. It was not active against the disease at the dosage tested and by the intraperitoneal (i.p.) treatment route employed in this study. The positive control, ribavirin, administered i.p. for 14 days at a dosage of 40 mg/ ml significantly increased the number of survivors. Three of 5 IT toxicity control animals developed some humoral response that inhibited PC4.9A6 binding to infected cells. They did not show any humoral response to the gelonin moiety of the IT.

No description available.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Bibliography: leaves 129-145.

<p>Radioactive substances are used <i>in vivo</i> to localize and characterize malignant tumours, generally by scintigraphic methods. In this context positron emission tomography (PET) in combination with radiolabelled monoclonal antibodies (mAbs) may provide a sensitive and specific method for detection of cancer. Individual dose calculations, based on such PET measurements, may be carried out to predict the possible use of mAbs labelled with therapeutic nuclides. The positron emitter ⁷⁶Br, with a half-life of 16 h, is a well-suited candidate for radiolabelling and PET imaging. One drawback of radiobromine is that bromide, the ultimate catabolite after degradation of brominated mAb, is only tardily excreted from the body and is evenly distributed throughout the extracellular space, thereby increasing the background radioactivity. The aim of this work was to produce ⁷⁶Br-mAb preparations with high accumulation and retention in tumour tissue together with a quick clearance of ⁷⁶Br-labelled catabolites. Furthermore, the possibility to use brominated or iodinated mAbs in combination with PET to predict ²¹¹At-mAb dosimetry was evaluated.</p><p>Monoclonal Abs directed against colorectal cancer were labelled with ⁷⁶Br using the direct Chloramine-T-method or indirectly by labelling the precursor molecule N-succinimidyl para-(tri-methylstannyl) benzoate with ⁷⁶Br, which was subsequently conjugated to the mAbs. Monoclonal Ab A33 labelled with ⁷⁶Br using the two labelling protocols was characterized in vitro and in vivo in a rat tumour xenograft model. The mAb A33 was also labelled with ¹²⁵I for comparison. In addition, mAb A33 was labelled with ²¹¹At, ¹²⁵I and ⁷⁶Br using the indirect labelling protocol and the mAb pharmacokinetics was studied in normal rats in order to estimate if data from brominated or iodinated mAb could be used for dosimetry of ²¹¹At in healthy organs and tissue.</p><p>In conclusion, both direct and indirect labelling resulted in high yields and mAbs with preserved immunoreactivity. <i>In vivo</i> characterization of ⁷⁶Br-brominated mAb A33 showed that the indirect labelling method makes ⁷⁶Br-brominated mAb A33 a promising candidate for tumour imaging with PET due to the faster excretion of radiolabelled catabolites compared with direct bromination. Finally, mAb A33 labelled with ⁷⁶Br and ^124/125I can be used to predict the ²¹¹At dose of astatinated mAb A33 in most organs given that a correction factor is applied for organs with varying uptake.</p>

Radioactive substances are used in vivo to localize and characterize malignant tumours, generally by scintigraphic methods. In this context positron emission tomography (PET) in combination with radiolabelled monoclonal antibodies (mAbs) may provide a sensitive and specific method for detection of cancer. Individual dose calculations, based on such PET measurements, may be carried out to predict the possible use of mAbs labelled with therapeutic nuclides. The positron emitter 76Br, with a half-life of 16 h, is a well-suited candidate for radiolabelling and PET imaging. One drawback of radiobromine is that bromide, the ultimate catabolite after degradation of brominated mAb, is only tardily excreted from the body and is evenly distributed throughout the extracellular space, thereby increasing the background radioactivity. The aim of this work was to produce 76Br-mAb preparations with high accumulation and retention in tumour tissue together with a quick clearance of 76Br-labelled catabolites. Furthermore, the possibility to use brominated or iodinated mAbs in combination with PET to predict 211At-mAb dosimetry was evaluated. Monoclonal Abs directed against colorectal cancer were labelled with 76Br using the direct Chloramine-T-method or indirectly by labelling the precursor molecule N-succinimidyl para-(tri-methylstannyl) benzoate with 76Br, which was subsequently conjugated to the mAbs. Monoclonal Ab A33 labelled with 76Br using the two labelling protocols was characterized in vitro and in vivo in a rat tumour xenograft model. The mAb A33 was also labelled with 125I for comparison. In addition, mAb A33 was labelled with 211At, 125I and 76Br using the indirect labelling protocol and the mAb pharmacokinetics was studied in normal rats in order to estimate if data from brominated or iodinated mAb could be used for dosimetry of 211At in healthy organs and tissue. In conclusion, both direct and indirect labelling resulted in high yields and mAbs with preserved immunoreactivity. In vivo characterization of 76Br-brominated mAb A33 showed that the indirect labelling method makes 76Br-brominated mAb A33 a promising candidate for tumour imaging with PET due to the faster excretion of radiolabelled catabolites compared with direct bromination. Finally, mAb A33 labelled with 76Br and 124/125I can be used to predict the 211At dose of astatinated mAb A33 in most organs given that a correction factor is applied for organs with varying uptake.