Affinity molecules can serve as precision tools for selective recognition and measurement of specific biomolecules in the fields of therapeutic drug monitoring and quality control in recombinant protein production. In therapeutic drug monitoring, affinity molecules can enable the accurate quantification of drug concentrations within physiological fluids, enhancing both the safety and efficacy of clinical treatments. In the realm of recombinant protein production, these molecules can allow precise isolation and measurement of desired recombinant proteins from complex mixtures by selectively targeting specific protein tags or domains, ensuring the consistency and purity of protein products. Currently, antibodies are most commonly used affinity reagents in these fields but are limited by production complexity, batch variability, high cost, and low stability. Aptamers, known as ‘chemical antibodies’ but composed of nucleotides, are considered potential next-generation affinity reagents. Aptamers are obtained via a synthetic process, termed SELEX, of iterative affinity selection and polymerase chain reaction (PCR) amplification of target-binding members from a randomized oligonucleotide library. This process is traditionally labor and resource-intensive and time-consuming. In this thesis, microfluidic technology is employed to enable time-efficient and cost-effective generation of aptamers for monoclonal antibodies and recombinant proteins toward applications in therapeutic drug monitoring and quality control of recombinant protein production. This thesis starts with a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection (called “chip-selection SELEX”), and fully integrated microfluidic affinity selection and PCR amplification (termed “full-chip SELEX”). The comparison results indicate that chip-selection SELEX offers the lowest cost and highest efficiency in aptamer isolation. We then use chip-selection SELEX to streamline the process of isolating anti-idiotype aptamers targeting human monoclonal antibodies against spike protein of SARS-CoV-2 virus. The process is completed within only 5 rounds of SELEX within two days, which represented a significant improvement when compared to conventional methods whose completion generally requires more than 10 SELEX rounds in up to a month. These anti-idiotype aptamers are combined with a graphene-based affinity nanosensor to enable rapid antibody concentration measurements to inform therapeutic decisions in a timely manner. In addition, a microfluidic dual-aptamer sandwich assay with highly efficient isolation of aptamers is developed to enable rapid and cost-effective detection of tag-fused recombinant proteins. This approach addresses both the limitations of current dual-aptamer assays and commonly encountered difficulties in the lack of aptamers available for such assays, by first using chip-selection SELEX to generate aptamers and then employing these aptamers to implement a microfluidic dual-aptamer assay for quality control during recombinant protein production. Despite the high efficiency in aptamer isolation using chip-selection SELEX, the full-chip SELEX platform is still desired for minimal manual operation and reagent consumption. The current full-chip SELEX platform has low isolation efficiency and could not offer information of affinity selection process. Herein, by introducing asymmetric PCR into the full-chip SELEX process, we improve the efficiency in aptamer isolation and can successfully monitor the selection progress. This real-time monitoring capability allows us to identify the optimal point to terminate the SELEX process, preventing the potential loss of aptamer candidates and reducing the overall consumption of time and reagents. In addition, introducing solution phase-based asymmetric PCR addresses a notable technical challenge of on-chip PCR bead replenishment, toward complete automation of the full-chip SELEX platform. Furthermore, a holder equipped with connection pins is designed to enable the reversible connection between gold electrodes and electrical wires. This design promotes the reusability of gold electrode-deposited glass substrates, resulting in a substantial reduction in chip fabrication costs. In addition to the SELEX protocol development effort, we also present efficient and cost-effective microfluidic approaches for post-SELEX aptamer characterization, including aptamer identification and kinetic aptamer-target binding measurements. To mitigate the expensive and time-consuming nature of aptamer identification from SELEX-generated target-binding sequence pools, we present an approach that is based on a cost-effective and efficient procedure to generate modified single-stranded DNA copies of the aptamer candidates and then assess the affinity of the resulting modified ssDNA strands to target molecules. The approach is applied to identify aptamers from 12 candidates with consistent results, but at a cost three times lower than that of established methods. We also present a microfluidic fluorescence assay, which exploits a synergistic combination of microfluidic technology and fluorescence microscopy, to realize cost-effective and multiplexed measurement of kinetics of aptamer-target analyte binding without requiring special-purpose equipment.

<p>Monoclonal antibodies (mAbs) as therapeutic proteins have shown great potential in treatment of various human diseases because of their highly specific nature. This has attracted worldwide attention leading to increased demand for such mAb products. To meet this demand large scale manufacturing is carried out using recombinant mammalian cell culture techniques for high yields and faster production. mAb products are worth the investment if produced in their native state. The quantity of mAb present in such cell cultures is very less and therefore special care is needed while handling them. Purifying antibody molecules from heterogeneous cell culture impurities and maintaining their native functional state is a critical task mainly because these antibodies are labile in nature. Care also need to be exercised during processing because mAbs have inherent tendancy to aggregate which is undesirable since such aggregates in antibody formulation produces immunogenic reaction when injected in humans. The other important factor in mAb purification is the processing cost involved since majority of the total production cost is utilized for purification of mAb. Protein-A chromatography is the first choice for purifying antibodies and is widely adopted. However failure in distinguishing between monomer and aggregate antibody molecules along with harsh acidic processing conditions necessitates the use of further purification steps.</p> <p>In this work various techniques for mAb processing are discussed and are outlined below:</p> <p>Removal of impurities from mAbs is a major challenge and this thesis discusses various processing options available to purify these mAbs. Impurities in mAb products are usually the aggregate byproducts formed due to unfolded monomer antibody molecules. These molecules are naturally hydrophobic in nature and display great differences in hydrophobicity on aggregation. Hydrophobic interaction membrane chromatography (HIMC) makes use of this hydrophobicity difference and helps in removal of aggregate impurities from monomer antibody.</p> <p>Heavy chain mAbs (hcmAbs) are promising new developments in the area of biopharmaceuticals because of their unique structural composition. Similar to conventional mAbs these hcmAbs are also rapidly finding their way into therapeutic markets. Purifying hcmAbs will be an important step in their development and for this purpose we use HIMC technique for removing impurities and obtain pure product.</p> <p>Antibody molecules are almost always lost as aggregates which leads to great economic losses and the ability to disaggregate these mAb oligomers would be of significant practical and scientific interest. In this work a novel thermalcycling technique is discussed to disaggregate such mAb oligomers and potentially recover functional monomer mAb molecules.</p> / Master of Applied Science (MASc)

Call number: LD2668 .T4 1985 G733 / Master of Science

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes, in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie, sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro- Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg- Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61 Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang. Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie, informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican; perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante. Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese differensiering en kanker, lê die grondslag vir verdere ondersoeke.

The development of recombinant proteins for therapeutic applications has revolutionized the pharmaceutical industry. In particular, monoclonal antibodies are the safest class of all therapeutic molecules and account for the majority of recombinant proteins currently undergoing clinical trials. A variety of technologies exist to engineer antibodies with a desired binding specificity and affinity, both of which are a prerequisite for therapeutic applications. This dissertation describes the implementation of a novel combinatorial library screening technology for the discovery and engineering of antibodies with unique binding properties. Combinatorial library screening technologies are used for the in vitro isolation of antibodies from large ensembles of proteins (libraries) typically produced by microorganisms using molecular biology techniques. Our lab has developed a powerful antibody discovery technology that relies on E. coli display by anchored periplasmic expression, otherwise known as APEx. First, I compared the effects of using combinatorial libraries comprising either smaller, monovalent single-chain antibody fragments (scFv), or the much larger, bifunctional full-length IgG antibodies. These technologies were used to isolate a small panel of antigen specific antibodies from the same library of antibody variable domains amplified from a mouse immunized with the Protective Antigen (PA) component from Bacillus anthracis, the causative agent of anthrax. Overall, IgG display resulted in the isolation of a broader panel of variable domain sequences. Most of these variable domains exhibited substantially reduced affinity when expressed as scFvs, which is consistent with the finding that none of these could be isolated from the equivalent scFv library. These results indicate that the antibody format used during in vitro selection affects which antibody variable domains will be discovered. Second, I developed several modifications of the APEx methodology to allow for more efficient recovery of antibodies with desired properties. Specifically, the system was reengineered to simultaneously account for antibody binding and expression levels in order to isolate the highest affinity antibodies with favorable expression characteristics. Third, the new approach, coupled with optimized fluorescence activated cell sorting (FACS) settings, was used to increase the affinity of an antibody by 35-fold resulting in a K[subscript D] of 100 pM. It was demonstrated that genetic transfer of this high affinity antibody specific for the V antigen of Yersinia pestis, the etiologic agent of the plague, conferred increased protection against intranasal challenge with a 363 LD₅₀ of Y. pestis in mice. / text

Cancer, historically considered a genetic disease, is currently acknowledged to affect the whole body. Our immune system is one key player that can elicit a response against malignant cells but can also promote tumorigenesis. Tumors avoid immune recognition by creating a suppressive microenvironment and inducing tolerance. T-cells are regarded a major effector cell type in tumor immunotherapy. An important ”switch” needed for T-cell activation involves so-called costimulatory and coinhibitory receptors. In this thesis, experimental tumor models were used to investigate the potential of immunomodulatory antibodies to stimulate immune cells and subsequently eliminate tumors. First, systemic antibody blockade of two negative checkpoint regulators (CTLA-4 and PD-1) present on T-cells was evaluated in combination with local CpG therapy or standard BCG treatment. Indeed, this combinatorial therapy with CpG augmented anti-tumor effects with increased levels of tumor-directed T-cells and reduced tumor-infiltrating Tregs. Secondly, as these immunomodulatory antibodies elicit severe side effects in patients, a local low-dose delivery regimen was explored as an alternative to systemic bolus treatment. Our results demonstrated that an approximately seven times lower dose of aCTLA-4, compared to systemic delivery, could eradicate both primary and distant tumors. CD40-expressing APCs are another potential target in antibody-mediated cancer therapy. CD40-stimulated dendritic cells (DCs) have the capability to activate tumor-directed T-cells to kill tumor cells. We next sought to investigate agonistic CD40 antibody efficacy and in vivo biodistribution when delivered locally compared to the equivalent systemic dose. Anti-tumor effects were dependent on CD8+ T-cells, host CD40 expression and the presence of tumor antigen at the injection site. CD40 antibodies were cleared from the circulation and accumulated in lymphoid organs, where, upon repeated aCD40 dosing, target APC populations increased in numbers and upregulated their surface CD40 expression. Lastly, CD40 agonist antibodies were mixed with nanoparticles to enhance their stimulatory properties. B-cells demonstrated increased proliferative capacity and DCs became more activated when exposed to the cocktail. Further, this combination reduced serum levels of pro-inflammatory cytokines compared to plain antibodies.       The results herein advocate further exploratory studies of the delivery of monoclonal antibodies at the tumor site in order to improve anti-tumor effects and reduce toxicity.

Nesse trabalho avaliamos a proteção que o anticorpo monoclonal (AcM) contra CMH e contra Hsp60 fornecem aos animais infectados com as leveduras de Paracoccidioides brasiliensis e P. lutzii em modelo profilático. O tratamento com os AcMs de isotipos IgG2a e IgG2b foram protetores, induzindo a secreção das citocinas IL-12p70, IFN-<font face=\"Symbol\">g e TNF-<font face=\"Symbol\">a, padrão de resposta imune Th1. Nós mostramos por imunomarcação que moléculas de CMH e Hsp60 estão acessíveis na célula. E o anticorpo monoespecífico contra CMH e os anticorpos policlonais contra melanina, gerados nesse trabalho foram eficazes nos ensaios in vitro de proteção. Foram gerados anticorpos monoclonais contra glicoproteina extraída de Pb18, o AcM reconheceu, por imunomarcação, estruturas internas e a parede celular da levedura. Avaliamos um novo modelo de hospedeiro, com o uso da larva Galleria mellonella, o que pode servir de triagem e reduzir desta forma o uso excessivo de camundongos, as leveduras P. lutzii e H. capsulatum são letais para as larvas e evocam resposta celular que se organizam semelhante a um granuloma. / In this work we evaluated the protection that the monoclonal antibody (MAb) against Hsp60 and against CMH provides the animals infected with the yeast Paracoccidioides brasiliensis and P. lutzii in prophylactic model. The treatment with the MAbs of IgG2a and IgG2b isotypes were protective, inducing the secretion of IL-12p70, IFN-<font face=\"Symbol\">g and TNF-<font face=\"Symbol\">a, Th1 pattern of immune response. We show by immunogold that Hsp60 and MHC molecules are accessible on the cell. And the monospecific antibody against HCM and polyclonal antibodies against melanin, generated in this study were effective in in vitro assays of protection. Monoclonal antibodies were generated glycoprotein extracted from Pb18, Mab recognized by immunogold, internal structures and cell wall material. Evaluated a new type of host, using the larvae Galleria mellonella, which can serve as a screening and reduce thus the overuse of mice, yeast P. lutzii and H. capsulatum are lethal to larvae and evoke cellular responses that are organized like a granuloma.

Anticorpos são moléculas de grande interesse científico e farmacêutico, principalmente, devido a sua alta especificidade contra antígenos determinados. Atualmente, anticorpos monoclonais estão entre os medicamentos (biofármacos) mais vendidos do mundo. São utilizados para o tratamento das mais diversas doenças, como câncer, retinopatias, doenças inflamatórias e do sistema imune, entre outras. Nos últimos 30 anos, as tecnologias para a obtenção de anticorpos monoclonais evoluíram muito, desde a tecnologia do hibridoma, até os processos de humanização de anticorpos murinos. Entre os métodos mais utilizados para a produção de anticorpos humanos, destaca-se a tecnologia do Phage Display. Nesta técnica, os genes que codificam as regiões variáveis de imunoglobulinas são inseridos no genoma de um bacteriófago, resultando na produção de partículas virais híbridas que contém fragmentos de anticorpos em fusão com uma das proteínas do capsídeo viral. Neste trabalho, desenvolvemos novos vetores para a apresentação de fragmentos ScFv em fusão com duas proteínas das proteínas do capsídeo viral, a pIII e pVIII. Os oligonucleotídeos utilizados para amplificar os genes de imunoglobulinas foram redesenhados e para minimizar a perda do repertório durante a produção da biblioteca, avaliamos em bancos de dados enzimas de restrição que não apresentam sítios de restrição nas sequencias gênicas. Esses sítios de restrição foram utilizados para construir as regiões de clonagem do vetor Phagemid. Outra etapa crítica na produção de bibliotecas de anticorpos é a reação do PCR de overlap, que pode restringir a diversidade de anticorpos e resultar na produção de amplicons codificando anticorpos truncados. Por isso, nossos vetores foram desenhados para permitir a clonagem direta das regiões variáveis das imunoglobulinas humanas ou murinas, sem a necessidade do PCR de overlap. Nossa expectativa, é que estes novos reagentes serão mais efetivos para a produção de novas bibliotecas de anticorpos pelo sistema do Phage Display. / Antibodies are molecules of great scientific and pharmaceutical interest, mainly because of their high specificity against certain antigens. Currently, monoclonal antibodies are among the best selling drugs (biopharmaceuticals) in the world. They are used for the treatment of the most diverse disorders, such as cancer, retinopathies, inflammatory and immune system diseases, among others. In the past 30 years, technologies for obtaining monoclonal antibodies has greatly evolved from hybridoma technology to the humanization processes of murine antibodies. Among the methods used for the production of human antibodies, the technology of Phage Display stands out. In this technique, the genes encoding the immunoglobulin variable regions are inserted into the genome of a bacteriophage, resulting in the production of hybrid virus particles which contain fragments of antibodies in fusion with one of the viral capsid proteins. In this work, we developed new vectors for the presentation of ScFv fragments in fusion with two proteins of viral capsid proteins, pIII and pVIII. The oligonucleotides used to amplify the immunoglobulin genes were redesigned and to minimize repertory loss during library production, we evaluated restriction enzymes in databases that lack restriction sites in the gene sequences. These restriction sites were used to construct the cloning regions of the Phagemid vector. Another critical step in the production of antibody libraries is the overlap PCR reaction, which may restrict the diversity of antibodies and result in the production of amplicons encoding truncated antibodies. Therefore, our vectors were designed to allow the direct cloning of human or murine Immunoglobulins variable regions without the need for overlap PCR. Our expectation is that these new reagents will be more effective for the production of new antibody libraries by the Phage Display system.

A partir de células de osteossarcoma humano (MG-63) que tem características de osteoblastos imaturos produzimos anticorpos monoclonais (Mabs) nomeados PSP 4-5, PSP 42-22 e PSP 85-9. Esses anticorpos reconhecem antígenos de 100, 26 e 20 kDa respectivamente. Avaliamos a especificidade dos mesmos, testando suas expressões em cortes congelados de tecidos oriundos da mesma célula mesenquimal, ou seja, osso, cartilagem, músculo cardíaco e tecido adiposo. Os anticorpos marcaram o periósteo, com distintos padrões de expressão, porém o PSP 42-22 foi o mais específico marcando a camada osteogênica do periósteo. Os anticorpos marcaram também células ósseas. Diante desses resultados nosso objetivo, no presente estudo, foi identificar os antígenos reconhecidos por esses anticorpos usando clonagem e sequenciamento dos genes em biblioteca de cDNA com capacidade de expressão proteica. Outro objetivo foi testar os anticorpos, pela técnica de imunohistoquímica (IHQ), em tumores ósseos primários visando estabelecer os padrões de marcação e se diferenciavam os diferentes tipos de tumores. Os antígenos reconhecidos pelos anticorpos PSP 42-22 e PSP 85-9 foram isolados, purificados e sequenciados. Devido ao elevado peso molecular do PSP 4-5 necessitaremos de outras técnicas para isolar o clone que codifica seu antígeno. O sequenciamento do clone isolado pelo PSP 42-22, mostrou uma sequência com 99% de homologia descrita no \"Homo sapiens\" como sendo \"serologically defined colon cancer antigen 3 (SDCCAG3), transcript variant 3\". A proteína SDCCAG3 foi descrita pela primeira vez em 1998 como um antígeno de câncer de cólon reconhecido por anticorpo autólogo. Vale lembrar que antígeno reconhecido pelo nosso anticorpo tem um peso molecular de aproximadamente 26 kDa, inferior ao da proteína SDCCAG3. Essa diferença poderia ocorrer devido a uma diferença no local da tradução do mRNA das células MG-63, produzindo uma proteína menor (isoforma), ou no clone isolado (3B2-3-1), poderia ocorrer uma mutação produzindo uma proteína mais curta que se expressaria na membrana celular ao contrário de uma proteína mais longa. Já o alinhamento das sequências obtidas dos clones isolados utilizando o PSP 85-9, mostrou uma sequência com 100% de concordância descrita com a porção não codante da proteína \"Homo sapiens microtubule associated protein, RP/EB family, member 1 (MAPRE1)\" portanto um falso positivo; já o clone 1A5-1-3, mostrou uma sequência de 99% de homologia com a proteína \"Homo sapiens Yip1 interacting factor homolog B (S. cerevisiae) (YIF1B), transcript variant 8\". Trata-se de uma proteína de membrana, com 6 isoformas diferentes que pode interagir com o receptor de serotonina. O resultado da IHQ nos tumores ósseos testados mostrou que o PSP 4-5 se expressou predominantemente no citoplasma de osteossarcoma, condrossarcoma e leiomiossarcoma e no núcleo de osteoblastoma. O anticorpo PSP 42-22 não reconheceu nenhum antígeno nos tumores avaliados. Quanto ao anticorpo PSP 85-9 nos condrossarcomas e leiomiossarcomas a expressão foi predominante citoplasmática e nos osteossarcomas e osteoblastoma foi nuclear. Em conclusão, identificamos os antígenos de dois dos anticorpos estudados que apresentam potencial diagnóstico diferencial em tumores ósseos primários / From human osteosarcoma cells (MG-63), which have characteristics of immature osteoblasts, we produced monoclonal antibodies (Mabs) named PSP 4-5, PSP 42-22 and PSP 85-9. These antibodies recognize antigens with 100, 26 and 20 kDa, respectively. We evaluated their specificity by testing their expression in frozen tissue sections from the same mesenchymal cells, that is, bone, cartilage, cardiac muscle and adipose tissue. The antibodies stained the periosteum, with distinct patterns of expression, but PSP 42-22 was the most specific, scoring the osteogenic layer of the periosteum. The antibodies also marked bone cells. With these results, our goal in this study was to identify the antigens recognized by these antibodies using cloning and sequencing of the genes in the cDNA library with capacity of protein expression. Another objective was to test the antibodies by immunohistochemistry (IHC) in primary bone tumors to establish standards for marking and whether they set apart different types of tumors. The antigens recognized by the PSP 42-22 and PSP 85-9 antibodies were isolated, purified and sequenced. Due to the high molecular weight of PSP 4-5 we will need other techniques to recognize its antigen. Sequencing of the clone isolated using the PSP 42-22 showed a sequence with 99% homology described in \"Homo sapiens\" as being \"serologically defined colon cancer antigen 3 (SDCCAG3), transcript variant 3\". The SDCCAG3 protein was first described in 1998 as a colon cancer antigen recognized by autologous antibody. It is worth remembering that the antigen recognized by our antibody has a molecular weight of approximately 26 kDa, lower than the SDCCAG3 protein. This difference could be due to a difference in mRNA translation site of MG-63 cells producing a lower protein; or on the isolated clone (3B2-3-1) a mutation could occur producing a shorter protein that expresses in the cell membrane instead of a longer protein. The alignment of the sequences obtained from isolated clones using the PSP 85-9 showed a sequence with 100% of homology described with the non-coding protein portion \"Homo sapiens microtubule associated protein, RP/EB family, member 1 (MAPRE1), mRNA\" therefore, a false positive; 1A5-1-3 clone showed a 99% homology sequence with the protein \"Homo sapiens Yip1 interacting factor homolog B (S. cerevisiae) (YIF1B), transcript variant 8, mRNA\". It is a membrane protein with 6 different isoforms which can interact with the serotonin receptor. The result of IHC in bone tumors tested showed that PSP 4-5 expressed predominantly in the cytoplasm of osteosarcoma, chondrosarcoma, leiomyosarcoma and osteoblastoma in was in nucleus. The PSP 42-22 antibody did not recognize any antigen in the tumors examined. As for the PSP 85-9 antibody in chondrosarcoma and leiomyosarcoma, the expression was predominantly cytoplasmic and the osteoblastoma was nuclear in osteosarcomas. In conclusion, we have identified the antigens of two of the antibodies studied which have potential differential diagnosis in primary bone tumors

La NADPH oxydase, Nox4, appartient à la famille des Nox qui génèrent les espèces radicalaires de l'oxygène, ROS, en transférant un électron à l'oxygène moléculaire. Malgré sa large distribution dans les tissus, Nox4 est encore mal comprise. Contrairement aux autres Nox, Nox4 est unique par son activité constitutive et sa capacité à former H2O2. Les ROS sont des espèces bactéricides dans les phagocytes et des outils de signalisation dans les cellules non phagocytaires en étant associés à de nombreuses pathologies inflammatoires et du vieillissement. Une étude de la structure en lien avec la fonction de Nox4 permettra de mettre l'accent sur un mécanisme de fonctionnement et sur de nouvelles cibles thérapeutiques. 5 nouveaux anticorps monoclonaux ont été générés contre une construction recombinante tronquée (AA: 206-578) de Nox4. La spécificité de 3 anticorps monoclonaux (8E9, 5F9, 6B11) a été confirmée par western blot dans les cellules HEK293 transfectées et le cortex de rein humain. L'anticorps 8E9 est le seul à permettre un marquage des cellules TRex-Nox4 sans perméabilisation par FACS. L'immunofluorescence confocale a montré que Nox4 est localisée dans la zone périnucléaire et le réticulum endoplasmique. La microscopie TIRF a confirmé sa présence dans la membrane plasmique. Un phénomène intéressant est que 5F9 ne détecte pas Nox4 à la membrane plasmique. L'épitope de 8E9 reconnaît une région sur la dernière boucle E extracellulaire de Nox4 (222H-E241), tandis que les anticorps monoclonaux, 6B11 et 5F9 marquent respectivement les régions 6B11 (389S-P416) et 5F9 (392D-F398). Par ailleurs, seuls 5F9 et 6B11 inhibent l'activité de Nox4, ce qui suggère que les deux régions marquées par ces ACm sont impliquées dans le transfert d'électrons. Une étude ciblée sur la boucle E de Nox4 a permis de montrer que le changement de 2 cystéines modifie la nature des ROS générés par Nox4 avec la production de O2- au lieu de H2O2. O2- est mis en évidence par la formation de peroxynitrite en présence de NO. Par ailleurs l'ACm 8E9 diminue la production de H2O2 dans les cellules COS7 qui expriment Nox4 à la membrane plasmique alors que celle de O2- est augmentée. Des constructions recombinantes de Nox4 (native ou tronquée) ont été générées par induction bactérienne, E.Coli, et par un système de transcription/traduction (RTS). Les protéines correspondantes, solubles, ont été produites à grande échelle et l'activité diaphorase mesurée; cette activité est constitutive. L'étude de la topologie membranaire de Nox4 et p22phox a été abordée en préparant des protéines de fusion avec l'ubiquitine marquée à la GFP. Cette méthode, TDUFA, particulièrement originale, devrait permettre d'appréhender la topologie de l'hétérodimère Nox4/p22phox, actif. / NADPH oxidase, Nox4, belongs to the Nox family which could generate reactive oxygen species by transferring an electron to molecular oxygen. Despite its wide distribution in tissues, Nox4 is still poorly understood. Unlike the other Noxes, Nox4 shows some unique characters: the constitutive activity, H2O2 formation. Nox4 involved ROS has been proposed to be implicated in several pathologies. Thus, to study the structure/function and the regulation of the activity of Nox4 will provide new ideas and new drug targets for the effective prevention and treatment of clinical diseases related with ROS. To know more about Nox4, in this study, 5 novel monoclonal antibodies were raised against a truncated recombinant protein (AA: 206-578) of Nox4. The specificity of 3 mAbs (8E9, 5F9, 6B11) was confirmed by western blot analysis in HEK293 transfected cells and human kidney cortex. In FACS studies, only mAb 8E9 could react with intact tet-induced T-RExTM Nox4 cells. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. An interesting phenomena is that mAb 5F9 failed to detect Nox4 at the plasma membrane. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4 (222H-E241), while mAb 6B11 (389S-P416) and 5F9 (392D-F398) are directed to its cytosolic tail. Cell-free oxidase assays showed a moderate but significant inhibition of constitutive Nox4 activity by mAb 5F9 and 6B11. To study the protein region which is responsible for the unique ability of Nox4 of releasing H2O2 rather than O2-, chimeric proteins and mutants were used. E-loop of Nox4 is 28 amino acid longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or change of the two cysteines in the E-loop switch Nox4 from H2O2 to O2- generation. In the presence of a NO donor, the O2--producing Nox4 mutants, but not widetype Nox4, generated peroxynitrite, excluding artifacts of the detection systems as the apparent origin of O2-. A second approach was used to confirm the responsibility of E-loop for the H2O2 formation. In Cos7 cells, which exhibit some plasma membrane expression of Nox4, addition of the mAb 8E9 decreased H2O2 production but increased O2- formation. Unlike Nox1 or Nox2, the E-loop of Nox4 contains a highly conserved histidine H222. Mutation of H222 also switched Nox4 from H2O2 to O2- formation. The structure of the E-loop might hinder O2- egress and/or provide a source for protons to accelerate dismutation to form H2O2. Two bacterial protein expression approaches (in vitro RTS and bacterial induction) were used to produce Nox4 cytosolic tail for characterizing the electronic transfer property of Nox4. The presence of rare codons (1363AGA AGA CUA1371) and high level of hydrophobicity affects the production of soluble and active recombinant Nox4Aqc and Nox4Bqc. After optimization of the conditions, soluble and active recombinant proteins were obtained by RTS or by bacteria induction. The soluble proteins were produced in large scale, purified onto affinity chromatography and were tested for the diaphorase activity (INT and cytochrome c). Results showed that electronic acceptor cytochrome c gives a higher rate than INT. Nox4Aqc produced a lower specific activity by a cell-based system compared to the protein synthesized in cell-free technology. This activity is not stimulated by the addition of cytosolic factors. A new method, topological determination by ubiquitin fusion assay (TDUFA), was used to investigate the topology of Nox4 and p22phox. ubGFP fusion proteins are used as tools to obtain details of membrane protein topology. This method was first validated by using two membrane proteins with known topology and then should get more topology information of Nox4 and p22phox further.