Monoclonal antibodies (mAbs) have become an important part of therapy for a number of cancers. The first mAb to be approved for clinical use is rituximab, which is currently used for the treatment of various B cell malignancies. Despite its clinical value, the mechanisms in which rituximab induces tumor regression are unclear. Growing evidence suggests that multiple mechanisms involving complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved. However, the direct interactions between CDC and ADCC have yet to be investigated. My studies examine the relationship between complement fixation and the activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model, and clinical samples from patients. Using these systems, I demonstrate that the initiation of the complement cascade inhibits NK cell activation and ADCC induced by rituximab in vitro. I also show that depletion of complement enhances the activation of NK cells and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that NK cell activation correlates with decreased complement activity in patients after rituximab treatment. The studies described in this dissertation have furthered the understanding of the mechanisms involved in antibody therapy. These results have described a novel inhibitory role for complement activity in the anti-tumor responses of mAbs. Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of complement may improve how current mAbs are used and the how mAbs are designed in the future.

<p>This study aims at introducing and describing a novel multi parameter analysis method to identify potential acquisition targets and to qualitatively and quantitatively evaluate the overall match between a target company and its acquirer. The method was tested with two recent real cases involving each an antibody based biotech company and a large fully integrated pharmaceutical company. The model was validated by comparing two independent antibody companies against the real cases, testing if they would have made better targets. It was found out that the in reality acquired companies scored highest, thus proving the validity of the method. One of the four potential targets got the highest scores for both acquirers. Consequently one of the acquired targets was only the second best match. The still independent companies would not have been better targets. The lowest scoring target company did get identical scores for both acquiring companies. Despite the proper prediction of targets, the scoring did not reveal the true underlying motives for the acquisitions, nor could significant parameters be identified to discriminate between target and non-target. This study adds a novel, valuable tool to the still limited arsenal of methods to qualitatively and quantitatively measure a match between target and acquirer solely based on publicly available data.</p>

<p>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.</p><p>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.</p><p>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.</p><p>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.</p>

This study aims at introducing and describing a novel multi parameter analysis method to identify potential acquisition targets and to qualitatively and quantitatively evaluate the overall match between a target company and its acquirer. The method was tested with two recent real cases involving each an antibody based biotech company and a large fully integrated pharmaceutical company. The model was validated by comparing two independent antibody companies against the real cases, testing if they would have made better targets. It was found out that the in reality acquired companies scored highest, thus proving the validity of the method. One of the four potential targets got the highest scores for both acquirers. Consequently one of the acquired targets was only the second best match. The still independent companies would not have been better targets. The lowest scoring target company did get identical scores for both acquiring companies. Despite the proper prediction of targets, the scoring did not reveal the true underlying motives for the acquisitions, nor could significant parameters be identified to discriminate between target and non-target. This study adds a novel, valuable tool to the still limited arsenal of methods to qualitatively and quantitatively measure a match between target and acquirer solely based on publicly available data.

Delivery of protein therapeutics is restricted to intravenous infusions due to protein-dependent problems including low solubilities, high viscosities, and physical instabilities. The ability to inject high concentrations of proteins via subcutaneous injections would increase accessibility and compliance. Large particles of a protein in a non-aqueous solvent can decrease the viscosity over a solution of equally concentrated individual protein molecules. The lower viscosity of a particle suspension is due to decreased surface area resulting in reduced electroviscous effects, solvation and deviations of the particle shape from a spherical geometry. Additional studies show that aqueous-based dispersions of antibody nanoclusters can be formed by increasing the attractive interactions between protein molecules using the excluded volume effects of extrinsic crowding agents. These novel, equilibrium, nanoclusters are maintained by a balance of highly attractive interactions and weak electrostatic repulsive interactions near the protein’s pI. These protein nanoclusters are ideal for subcutaneous delivery as they have low interactions between the colloids, are reversible in nature, and dissolve rapidly upon dilution in a buffer media. Through in vivo mouse studies, the bioavailability of a monoclonal antibody in the dispersion is prolonged and higher doses can be administered versus a solution. Overall, these studies with high concentration, low viscosity subcutaneous injections of protein therapeutics open new opportunities in biotechnology. For oral delivery of itraconzole, controlled flocculation of individual polymerically-stabilized nanoparticles is used to increase supersaturation. Flocculation of these nanoparticles is achieved by desolvating the polymer by changing the pH. The flocculated dispersions can then be easily filtered. The final amorphous powder maintains high supersaturation with simulated stomach and small intestine conditions and improves bioavailability of itraconazole, over the commercial product, Sporanox®. / text

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The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25 mM) of cultures established at high density (>106 cells/ml) over 24 h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24 h resulted in a 52% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI), which showed a direct correlation to the cell specific glucose uptake. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.

Islet transplantation is a more physiological way to treat type 1 diabetes. However, shortage of donor tissue and chronic administration of immune suppressive drugs has limited the widespread application of this therapy for all patients with type 1 diabetes, particularly children suffering from this disease. Xenogeneic islet transplantation particularly neonatal porcine islets (NPI) holds promise for clinical transplantation because of the potentially unlimited supply of islets. New evidence suggests that monoclonal antibodies (mAbs) specific for immune cell surface molecules could be employed in the prevention of islet graft rejection as well as induction of immunological tolerance to the transplanted grafts without the need for continuous administration of harmful immune suppressive drugs. It was shown by our group that short-term administrations of a combination of anti-LFA-1 and anti-CD154 mAbs which targets both adhesion and costimulatory pathways of T cell activation, is highly effective in preventing NPI xenograft rejection. In this thesis, we determined whether short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs could induce tolerance to NPI xenografts. Our data show that this combination of mAbs can induce dominant, species and tissue specific tolerance to NPI xenografts which is mediated by regulatory T cells in non-autoimmune prone B6 mice. We also found that T cell subsets such as CD4+ and CD8+ T cells as well as antigen presenting cells (APC) play an important role in the induction and maintenance of tolerance to NPI xenografts. In addition we found that PD-1/PDL interaction is important for induction and maintenance of tolerance to NPI xenografts. Finally, we found that this combined mAb therapy was effective in preventing NPI xenografts rejection in autoimmune prone NOD mice when it was combined with anti-CD4 mAb. It is may hope that the research presented in this thesis will provide insight into the nature of the immune responses to xenogeneic islet transplantation in humans and aid in the development of effective, tolerance inducing therapies, so that patients with T1DM will once again know a life free from their disease. / Experimental Surgery

Orientador: Valéria Marçal Félix Lima / Banca:Gisele Fabrino Machado / Banca:Sandra Helena Penha de Oliveira / Resumo: A leishmaniose é uma doença imunossupressora causada por protozoários do gênero Leishmania, tendo os cães como reservatório doméstico. A molécula Programmed Cell Death 1 (PD-1) é altamente expressa em células leucocitárias de cães com leishmaniose visceral e promove a exaustão dos linfócitos T e a supressão da secreção de citocinas. Como o PD-1 tem função supressiva em relação à imunidade celular, avaliamos o efeito de anticorpos bloqueadores de PD-1 na produção de NO, ROS, interleucina 17 (IL-17) e na carga parasitária em cultura de leucócitos esplênicos de cães naturalmente infectados. In vitro, o bloqueio de PD-1 promoveu aumento dos níveis de NO intracelular e reduziu os de IL-17 no sobrenadante da cultura, além de reduzir a carga parasitária, más não alterou os níveis de ROS. Concluímos que a PD-1 participa na regulação da resposta imune e que o anticorpo bloqueador é efetivo na restauração da atividade microbicida do hospedeiro. Isso pode ser investigado em estudos imuno-terapêuticos no futuro. / Abstract: Leishmaniasis is an immunosuppressive disease caused by protozoa of the genus Leishmania for which dogs are the domestic reservoir. The programmed cell death 1 molecule (PD-1) is highly expressed in leukocyte cells of dogs with visceral leishmaniasis, and it promotes T lymphocyte exhaustion and suppression of cytokine secretion. Because PD-1 has a suppressive function regarding cell immunity, we evaluated the effect of PD-1 blocking antibodies on NO, ROS and interleukin 17 (IL-17) production and on parasite load in spleen leukocyte cultures from naturally infected dogs. In vitro, PD-1 blocking promoted increased levels of intracellular NO and reduced the levels of IL-17 in the culture supernatant, in addition to reducing the parasite load, but do not change ROS levels. We conclude that PD-1 participates in regulation of the immune response and that the blocking antibody is effective in restoring host microbicidal activity. This can be investigated in an immuno-therapeutic study in the future. / Mestre

Submitted by Gabriela Torres Rebech (gtr.torres@hotmail.com) on 2018-05-11T23:25:50Z No. of bitstreams: 1 Dissertação Gabriela.pdf: 515150 bytes, checksum: 8dcf7e5ca4b514e5c7c8ada92438306b (MD5) / Approved for entry into archive by Ederson Vasconcelos Pereira null (edersonpereira@fmva.unesp.br) on 2018-05-14T17:16:11Z (GMT) No. of bitstreams: 1 rebech_gt_me_araca_int.pdf: 515150 bytes, checksum: 8dcf7e5ca4b514e5c7c8ada92438306b (MD5) / Made available in DSpace on 2018-05-14T17:16:11Z (GMT). No. of bitstreams: 1 rebech_gt_me_araca_int.pdf: 515150 bytes, checksum: 8dcf7e5ca4b514e5c7c8ada92438306b (MD5) Previous issue date: 2018-04-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose é uma doença imunossupressora causada por protozoários do gênero Leishmania, tendo os cães como reservatório doméstico. A molécula Programmed Cell Death 1 (PD-1) é altamente expressa em células leucocitárias de cães com leishmaniose visceral e promove a exaustão dos linfócitos T e a supressão da secreção de citocinas. Como o PD-1 tem função supressiva em relação à imunidade celular, avaliamos o efeito de anticorpos bloqueadores de PD-1 na produção de NO, ROS, interleucina 17 (IL-17) e na carga parasitária em cultura de leucócitos esplênicos de cães naturalmente infectados. In vitro, o bloqueio de PD-1 promoveu aumento dos níveis de NO intracelular e reduziu os de IL-17 no sobrenadante da cultura, além de reduzir a carga parasitária, más não alterou os níveis de ROS. Concluímos que a PD-1 participa na regulação da resposta imune e que o anticorpo bloqueador é efetivo na restauração da atividade microbicida do hospedeiro. Isso pode ser investigado em estudos imuno-terapêuticos no futuro. / Leishmaniasis is an immunosuppressive disease caused by protozoa of the genus Leishmania for which dogs are the domestic reservoir. The programmed cell death 1 molecule (PD-1) is highly expressed in leukocyte cells of dogs with visceral leishmaniasis, and it promotes T lymphocyte exhaustion and suppression of cytokine secretion. Because PD-1 has a suppressive function regarding cell immunity, we evaluated the effect of PD-1 blocking antibodies on NO, ROS and interleukin 17 (IL-17) production and on parasite load in spleen leukocyte cultures from naturally infected dogs. In vitro, PD-1 blocking promoted increased levels of intracellular NO and reduced the levels of IL-17 in the culture supernatant, in addition to reducing the parasite load, but do not change ROS levels. We conclude that PD-1 participates in regulation of the immune response and that the blocking antibody is effective in restoring host microbicidal activity. This can be investigated in an immuno-therapeutic study in the future.