Made available in DSpace on 2014-08-20T13:33:01Z (GMT). No. of bitstreams: 1 tese_flavia_aleixo_vasconcellos.pdf: 1086060 bytes, checksum: 69a51e8723d756b44009a0d489d3f4d5 (MD5) Previous issue date: 2011-12-01 / Malignant neoplasms such as breast cancer deserve attention for its high prevalence and great demand of financial resources, representing a major problem of public health all over the world. In Brazil, mortality rates from breast cancer remain high, most likely because the disease is diagnosed in advanced stages. Tumor markers can be useful in diagnosis, staging and in the evaluations of therapeutic responsiveness and disease prognostic. The overexpression of the oncogene Human Epidermal growth factor receptor 2 (HER2), a prognostic tumor marker in breast cancer, is observed in 20-30% of breast cancers in humans and canines. In this context, this study reports the development of new murine monoclonal antibodies from a recombinant protein corresponding to the extracellular portion of the HER2 receptor for use in immunohistochemistry (IHC) and in the development of other immunoassays. These antibodies were produced, characterized and tested subsequently in histological sections of human and canine breast tumors. Among five hybridomas produced, two demonstrated potential for use in immunohistochemistry of these two tumors. / As neoplasias malignas, como o câncer de mama, atraem a atenção em todo o mundo por sua alta prevalência e grande demanda de recursos financeiros, e por representarem um grande ônus social. No Brasil, as taxas de mortalidade por câncer de mama continuam elevadas, muito provavelmente porque a doença ainda é diagnosticada em estágios avançados. Marcadores tumorais podem ser úteis no diagnóstico, no estadiamento e nas avaliações da resposta terapêutica e do prognóstico da doença. A superexpressão do oncogene Human Epidermal growth factor Receptor 2 (HER2), um marcador de prognóstico em câncer de mama, é evidenciada em 20-30% dos tumores mamários em humanos e caninos. Nesse contexto, o presente trabalho relata o desenvolvimento de novos anticorpos monoclonais murinos apartir de uma proteína recombinante correspondente à porção extracelular do receptor HER2 para uso em técnica de imunohistoquímica (IHQ) e desenvolvimento de outros ensaios imunodiagnósticos. Estes anticorpos monoclonais foram produzidos, caracterizados e posteriormente testados em cortes histológicos de tumores mamários humano e canino. Entre cinco hibridomas produzidos, dois deles demonstraram potencial para uso na técnica de imunohistoquímica nestes tumores.

Background. Immunohistochemistry is a useful method in the differential diagnosis between pleural mesotheliomas and metastatic adenocarcinomas in the pleura. Cytokeratin 20 and 7 have been used successfully as markers in studies determining primary location of adenocarcinomas from metastases. The current study is a complementary research of archived paraffininbedded material of cases with cancer origin. This study contributes a bigger statistical material that may facilitate the search for unknown primary site of adenocarcinoma by identification of metastatic cells in the pleura. Methods. Cells from the pleura taken from fifteen patients with diagnosed cancer of different types and eleven patients with cancer of unknown origin, were stained with antibodies against the tumour markers: Ber EP 4, calretinin, cytokeratin 20 and 7, estrogen receptor α, thyroid transcription factor, prostate-specific antigen and Cdx2.The staining was conducted in an automated immunohistochemical system. The staining of each kind of antibody was confirmed by a control section staining. Results. All control staining ended perfect The whole panel of antibodies used on mammary cancer showed the same pattern for every antibody. Of the patients with cancer of unknown origin there were four that gave the same pattern, two men and two women. The women are deceased. To make a more careful evaluation more information and clinic background is needed. The number of samples is too small to draw any statistical conclusions. Comment. Although the control staining was perfect the negative result of CK20 in the cases of diagnosed colon cancer was unexpected. This staining should be performed again to confirm the result. In some cases the number of cells were to few for a certain evaluation. The slides and the results of this work will be archived for further research.

La transformation des cellules normales de l’organisme en un phénotype malin est souvent accompagnée de changements dans leur antigénicité. L’antigène Tn (GalNac-O-Ser/Thréo) est un antigène (Ag) glycopeptidique spécifique des tumeurs et exprimé à la membrane plasmique des cellules cancéreuses dans la majorité des carcinomes humains ainsi que dans certaines tumeurs hématologiques, tandis qu’il n’est pas détecté dans les cellules normales. Il représente donc une cible potentielle très intéressante pour l’immunothérapie passive par anticorps, car il n’est pas détectable dans les cellules normales, mais est démasqué dans environ 90% des cancers épithéliaux du fait d’une dérégulation des processus de glycosylation. Les anticorps monoclonaux (AcM) spécifiques d’antigènes exprimés à la membrane des cellules tumorales ont une efficacité prouvée dans le traitement de certains cancers. Ces AcM thérapeutiques sont particulièrement intéressants pour le traitement des cancers du fait de leur forte spécificité pour les cellules tumorales et de leur faible toxicité pour les cellules normales, contrairement aux chimiothérapies conventionnelles, mais leur mécanisme d’action est encore mal connu. L’AcM Chi-Tn est un anticorps chimérique homme/souris capable de se fixer de façon spécifique à l’antigène tumoral Tn, alors qu’il ne se fixe pas sur les cellules normales. Cet AcM pourrait donc être envisagé comme agent thérapeutique dans le traitement des cancers épithéliaux par immunothérapie passive.Nous nous sommes intéressés à l’AcM Chi-Tn non couplé en vue d’analyser son mécanisme d’action et d’évaluer son efficacité thérapeutique in vivo. Nous avons montré que l’AcM Chi-Tn seul ne possède pas d’effet toxique direct sur les lignées de cellules tumorales Tn-positives in vitro. Cependant, en présence de macrophages, cet AcM est capable d’induire la lyse de ces cellules par un mécanisme d’ADCC. In vivo, l’AcM Chi-Tn, associé à la cyclophosphamide, induit le rejet d'une tumeur du sein dans plus de 80% des souris. Cette inhibition de la croissance tumorale est abolie chez les souris déficientes pour la chaine associée aux récepteurs RFc activateurs, suggérant in vivo un mécanisme d’ADCC. Par l’étude microscopique du microenvironnement tumoral, nous avons observé que les cellules tumorales forment in vivo des synapses avec des macrophages, des neutrophiles, mais aussi des lymphocytes B. Des expériences de survie in vivo chez des souris déficientes pour différentes populations cellulaires montrent que les lymphocytes T semblent nécessaires à la protection des souris par Chi-Tn contre la tumeur. Ainsi, ces résultats confirment le rôle des effecteurs exprimant des RFc activateurs, mais aussi le rôle indispensable de la réponse immune adaptative pour assurer l'effet thérapeutique des AcM.Nous nous sommes également intéressés à l’utilisation potentielle de l’AcM Chi-Tn comme vecteur d’agents cytotoxiques. In vivo, dans un modèle de tumeurs solides chez la souris, des expériences de biodistribution montrent que l’AcM Chi-Tn est capable de cibler spécifiquement les zones tumorales, ce qui en fait un anticorps potentiellement utilisable comme vecteur de molécules toxiques. L’internalisation du complexe anticorps/antigène cible est un pré-requis nécessaire à l’utilisation de l’anticorps conjugué. Nous avons montré in vitro que l’AcM Chi-Tn est internalisé dans les endosomes précoces et de recyclage pendant un temps relativement long, faisant de cet AcM un bon candidat pour être couplé à des agents cytotoxiques. Durant ma thèse, nous avons couplé l’AcM Chi-Tn à la toxine saporine ou à la molécule cytotoxique auristatine F, et nous avons montré in vitro que ces conjugués sont cytotoxiques sur des lignées cellulaires Tn-positives. / Pas de résumé en anglais

Radioimmunotherapy (RIT) and radioimmunolocalisation (RIL) are developing and promising technologies to diagnose and treat tumours by use of radiolabelled antibodies targeting tumour specific antigens. The major reason why RIL and RIT not are efficient enough, is the comparatively low accumulation of radiolabelled antibodies in the tumours. Irrespective of the antigen - antibody system used, the maximal tumour uptake in humans is often limited to below 0.1 % of the total injected dose, with significant radionuclide remaining in the blood pool and extravascular fluid. In the present thesis, the following putative improvement techniques for radioimmunotargeting have been evaluated in an experimental model using HeLa cell-xenografted nude mice: 1) Repetitive, simultaneous targeting of different antigens, 2) Removal of non-targeting antibodies using secondary antiidiotypic antibodies, 3) Preinjection of unlabelled antibody to remove shedded antigen and 4) Use of fractionated antibody administration. By use of multiple injections of mixtures of two different 131I-labelled monoclonal antibodies targeting placental alkaline phosphatase (H7) and cytokeratin 8 (TS1), respectively, a significant tumour growth inhibition compared to controls, was obtained. In the treated group, a negligible increase in tumour volume was seen compared to the control group, in which a 20-fold increase was observed. Quantitative determinations of volume densities of viable tumour cells, necrotic cells and connective tissue demonstrated no significant differences in the relative proportions between the groups, indicating that the irradiation caused decelerated growth. Using hybridoma technology, monoclonal antiidiotypic antibodies were generated against both TS1 and H7. The in vitro and in vivo effects of these antibodies, aH7 and aTSl, were investigated. Both these antiidiotypes were found to generate stable complexes with the radiolabelled idiotypic antibody, as revealed by gel-electrophoresis and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate-, dissociation rate-, and affinity constants between the reactants were determined. In vivo, the antiidiotypes promoted a rapid dose dependent clearance of the 125I-labelled idiotypes with a decrease in total body radioactivity and concomitant dramatic increase in non-protein bound 125I excreted in the urine. The syngeneic monoclonal antiidiotypic antibody αTSl, was furthermore evaluated as a secondary clearing antibody at radioimmunolocalisation. Injection of αTSl in a molar ratio of 0.5-0.75:1 to TS1, 24 hours after the 125I-labelled TS1 improved the tumour to normal tissue ratio 2-3 fold. This was due to a decreased level of total body radioactivity as well as a slight decrease in tumour-radioactivity. A model describing the kinetics of the involved components, i.e. the antigen, the idiotype and the antiidiotype was presented. It is concluded that high affinity monoclonal antiidiotypes can be used as tools to regulate the levels of idiotypic antibodies in vivo. This strategy, combined with preinjection of non­labelled idiotypic antibodies, caused accumulated doses of 3 Gy to the tumour and 0.9 Gy to non tumour tissues as calculated for 125I-labelled antibodies (80 MBq/mg) by MIRD formalism based on repetitive quantitative radioimmunoscintigraphies. By approaching the maximal tolerated whole body radiation dose for mice (i.e. 6 Gy), it can be estimated that doses up to 20 Gy are possible to obtain following one single injection of labelled antibody. It was furthermore demonstrated that a single bolus injection of antibody is to be preferred, compared to exactly the same dose divided into three or ten fractions. Thus, not only the dose of radioactivity, but also the amount of antibody should be considered for fractionated RIT. In summary, the thesis demonstrates that several techniques can be used to improve radioimmunolocalisation and to approach the proposed 70 Gy required to sterilise tumours at radioimmunotherapy. / digitalisering@umu.se

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21817号 / 農博第2330号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5189(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 間藤 徹, 教授 髙部 圭司, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Melanoma is one of the most aggressive and deadly forms of skin cancer. Arising from melanocytes, a pigment cell population derived from the neural crest, melanomas often adopt characteristics associated with the neural crest – the ability to rapidly proliferate, migrate and invade throughout the body. Historically, these characteristics along with a baseline resistance to chemotherapy have made melanoma extremely difficult to treat. Improvements in targeted and immunotherapeutic options have improved patient outcomes, but many patients still experience limited durable responses to therapy. In order to improve patient outcomes, new potential avenues of therapy must be identified based on the underlying pathogenesis of the disease. We previously identified and characterized the function of a novel melanoma oncogene, GDF6, uncovering a role in promoting melanoma cell survival and dedifferentiation by activating a neural crest identity. Here, we have a) identified a role for GDF6-activated BMP signaling during melanocyte development that forms a basis for its oncogenic role in melanoma, b) determined BMP signaling may play a role in promoting a neural crest-like state during melanoma initiation, and c) assayed novel monoclonal antibodies targeting GDF6 for use as blocking antibodies to treat advanced melanoma. Previous work identified GDF6 as a melanoma oncogene that promotes melanoma progression through suppression of apoptosis and differentiation in melanoma cells, by regulating neural crest factor expression and neural crest identity, suggesting a potential role for GDF6 in the embryonic neural crest. Additional studies had previously identified roles for GDF6 and its orthologous genes in specific biological contexts, including embryonic neuronal cell survival, bone and cartilage development, embryonic eye development, and bone and ligament repair in adult tissue. Furthermore, a study had indicated a role for a GDF6 ortholog, gdf6a, during zebrafish neural crest induction, but had not uncovered any specific role for gdf6a in further development of the neural crest or in any neural crest derivatives. We determined blocking gdf6a-activated BMP signaling acts to increase melanocyte development during embryogenesis by increasing the proportion of neural crest cells activating the pigment cell marker, mitfa. Furthermore, we showed the increase in melanocytes is at the expense of the iridophore population. These results indicate GDF6 function in melanoma is a reiteration of the normal physiological function of GDF6 during embryonic melanocyte development from the neural crest. Given these results and our previous findings of the role of GDF6-activated BMP signaling established melanomas, we hypothesized a potential role for GDF6-activated BMP signaling during melanoma initiation. Previous studies have determined neural crest identity and neural crest-like characteristics to be crucial during multiple phases of melanoma, including initiation, progression, and metastasis. We evaluated melanoma initiating lesions to determine the potential impact of BMP signaling on development and progression of these lesions. We found early lesions in our model to have active BMP signaling and that modulation of BMP signaling could alter the rate of development of these lesions in our animals. Furthermore, BMP modulation ultimately impacted the development of these lesions into melanomas. Together, these results indicate BMP signaling is a potential driving pathway during melanoma initiation and progression. Finally, we wanted to determine the therapeutic potential of targeting GDF6 in order to treat patients with advanced melanoma. Given our previous findings and mechanism of ligand-activated BMP signaling, we hypothesized a monoclonal antibody targeting GDF6 could block GDF6 activity at its receptor on melanoma cells, thus inhibiting GDF6-activated BMP signaling. Monoclonal antibodies have been widely used as therapy in cancer as well as many other rheumatologic and immunologic conditions. We established a panel of GDF6-targeting antibodies via a hybridoma approach. We then assessed the antibodies ability to identify mammalian GDF6 in vitro and performed functional assays to determine if anti-GDF6 antibody treatment yielded the expected results of inhibiting GDF6-activated BMP signaling. We observed decreased pathway activity, decreased cell viability, and increased cell death in melanoma cells treated with anti-GDF6 antibodies in vitro. We further investigated whether these antibodies could exert anti-melanoma effects in vivo. Together, these results indicate potential therapeutic value for our antibodies in treating GDF6-positive melanomas.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Klaudia Sabolová Supervisor: Mgr. Pavel Bárta, Ph.D. Title of diploma thesis: The preparation and the following in vitro saturation study of the radiopharmaceutical 99m Tc-DTPA-ramucirumab on PC-3 cell line In cancer treatment, immunology is given prominence, which compared with chemotherapy and radiotherapy has a lower risk of side effects on healthy tissues. Immunotherapy includes application of monoclonal antibodies aimed at some tumour antigens using either non conjugated monoclonal antibodies or conjugated ones with an appropriate effector element, such as radionuclide. Angiogenesis plays the important role in pathogenesis of tumour diseases. Angiogenic process is regulated mostly by the interactions among vascular growth factors (VEGFs) and VEGF receptors (VEGFR). The main regulator of angiogenesis is VEGF-A. The blocking of the interaction among VEGF-A and its receptors VEGFR-1 and VEGFR-2 inhibits angiogenesis, and so does the growth of tumours. Ramucirumab is the monoclonal antibody with antiangiogenic effect, which blocks this interaction by its binding to the extracellular VEGFR-2 domain with high affinity. The presented study is focused on ramucirumab...

The extracellular matrix (ECM) of the skeletal muscle provides the framework for the muscle structure and plays a key role in the repair and maintenance of myofibers through the resident fibroblasts and muscle satellite cells. However, excessive production of ECM components, notably collagen, leads to fibrosis which impedes muscle function, impairs the natural repair process, and leads to muscle weakness. Fibrosis is a hallmark of muscular dystrophies, including Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy is a terminal, x-linked disorder characterized by progressive muscle wasting as muscle fibers are replaced by fibrosis and fat. There are approximately 300,000 DMD patients worldwide, and the few disease modifying treatments are genotype specific, only helping a small percentage of the patient population. Myostatin is a member of the transforming growth factor beta (TGF-β) family of ligands, is a negative regulator of muscle mass, and may also contribute to the fibrotic environment in dystrophic muscle through myofibroblast proliferation and survival. Therefore, myostatin blockade could potentially ameliorate muscle weakness in DMD patients by increasing skeletal mass and function while also reducing the accumulation of fibrosis. A murine anti-myostatin antibody, mRK35, and its humanized analogue, domagrozumab, are specific and potent inhibitors of myostatin. mRK35 was tested in multiple mouse models, from healthy C57Bl/6 and C57Bl/10, mildly dystrophic C57Bl/10.mdx, and severely dystrophic D2.mdx mice, for changes in muscle mass, muscle function, and fibrotic content. Additionally, inflammatory, fibrotic, and myogenic gene expression changes were analyzed in the severely dystrophic animals treated with mRK35. Domagrozumab was tested in non-human primates (NHPs) for changes in skeletal muscle mass. Myostatin blockade with mRK35 resulted in muscle anabolic and functional improvements in healthy murine models and NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume. However, as mice age or as the dystrophic severity of the model increases, the anabolic effect of myostatin inhibition is diminished. The extensor digitorum longus (EDL) muscle escapes this trend and is the most responsive to myostatin inhibition across all mouse strains and disease severities. However, analysis of the fibrotic content in the triceps and diaphragms of D2.mdx mice treated with mRK35 for 8 weeks does not reveal any change in fibrotic content. Gene expression changes in the muscles within these mice appear to be tightly tied to their healthy or dystrophic state and myostatin inhibition has minimal effect. In sum, while specific myostatin inhibition with mRK35 increases muscle weight and function in mice, there is no conclusive evidence of reduced muscle fibrosis. / 2023-06-22T00:00:00Z

Following rabies virus (RABV) exposure, a combination of thorough wound washing, multiple-dose vaccine administration and the local infiltration of rabies immune globulin (RIG) are essential components of modern post-exposure prophylaxis (PEP). Although modern cell-culture-based rabies vaccines are increasingly used in many countries, RIG is much less available. The prohibitive cost of polyclonal serum RIG products has prompted a search for alternatives and design of anti-RABV monoclonal antibodies (MAbs) that can be manufactured on a large scale with a consistent potency and lower production costs. Robust in vitro neutralization activity has been demonstrated for the CL184 MAb cocktail, a 1:1 protein mixture of two human anti-RABV MAbs (CR57/CR4098), against a large panel of RABV isolates. In this study, we used a hamster model to evaluate the efficacy of experimental PEP against a lethal challenge. Various doses of CL184 and commercial rabies vaccine were assessed for the ability to protect against lethal infection with representatives of four distinct bat RABV lineages of public health relevance: silver-haired bat (Ln RABV); western canyon bat (Ph RABV); big brown bat (Ef-w1 RABV) and Mexican free-tailed bat RABV (Tb RABV). 42–100% of animals survived bat RABV infection when CL184 (in combination with the vaccine) was administered. A dose-response relationship was observed with decreasing doses of CL184 resulting in increasing mortality. Importantly, CL184 was highly effective in neutralizing and clearing Ph RABV in vivo, even though CR4098 does not neutralize this virus in vitro. By comparison, 19–95% survivorship was observed if human RIG (20 IU/kg) and vaccine were used following challenge with different bat viruses. Based on our results, CL184 represents an efficacious alternative for RIG. Both large-scale and lower cost production could ensure better availability and affordability of this critical life-saving biologic in rabies enzootic countries and as such, significantly contribute to the reduction of human rabies deaths globally.

Despite the progress made in cancer research, cancer remains one of the leading causes of death worldwide. Although the development of new cancer treatments has improved cancer patients' survival rate, a significant number of patients experience refractory and recurrence events with serious side effects. It is known that the immune system actively participates in eliminating cancer. However, cancer cells can develop mechanisms to evade the immune system resulting in immunotolerance. Immunotherapy aids the patient's immune system's ability to recognize and eliminate cancer cells. During the last three decades, immunotherapy has gradually emerged as an effective and more specific approach to treat cancer. Particularly monoclonal antibodies and adoptive cell therapies such as chimeric antigen receptor (CAR) T-cells have proven highly effective. Nevertheless, the success of these novel therapies depends on discovering suitable tumor targets. Recently, we reported localization of Thymidine Kinase 1 (TK1) to the plasma membrane of certain cancer cells but have not found such localization on normal cells. Similarly, another nucleotide salvage pathway enzyme Hypoxanthine Guanine Phosphoribosyltransferase (HPRT), has also been reported to be localized to the plasma membrane of certain cancer cells. Thus, TK1 and HPRT membrane-associated forms can be potential tumor targets for cancer immunotherapy. This dissertation describes the immunotargeting of TK1 for the selective elimination of tumor cells and the surface localization of HPRT on the plasma membrane of cancer cells. Using hybridoma and phage display technologies, we developed monoclonal antibodies (mAb) and isolated human single domain antibodies (sdAb) specific to human TK1. We confirmed that antibodies and sdAbs could target TK1 on the plasma membrane of lung, breast and colon cancer cells, but not on healthy cells. In addition, we demonstrated that cancer cells expressing membrane-associated TK1 (mTK1) co-cultured with human mononuclear cells (MNC) were selectively eliminated through antibody-dependent cell-mediated cytotoxicity (ADCC) when anti-TK1 mAbs were added. Furthermore, we designed novel TK1 specific tumor targeting receptors and expressed them in human T cells and human macrophages. Finally, we proposed using both TK1 and HPRT as biomarkers for the early detection and monitoring of follicular lymphoma (FL), a disease that is usually detected at advanced stages. The knowledge generated from the data presented in this dissertation indicates that TK1 and HPRT may be suitable immunotherapeutic targets for antibody-based and adoptive cell-based therapies against both liquid and solid malignancies. It also proposes the incorporation of TK1 and HPRT as molecular biomarkers for the early detection and monitoring of FL.