Τα MHC τάξης I πεπτίδια περιορισμένα μόνο σε καρκινικό ιστό, τα οποία παράγονται από πρωτεΐνες του ίδιου του οργανισμού υπερεκφραζόμενες από μία μεγάλη ποικιλία ανθρώπινων κυττάρων νεοπλασιών, είναι δυνητικοί στόχοι για CTL (κυτταροτοξικά Τ λεμφοκύτταρα - Cytotoxic T lymphocyte), στα οποία βασίζεται η ανοσοθεραπεία πολλών ειδών καρκίνου. Ωστόσο, δεδομένου ότι παρουσιάζονται και από τα φυσιολογικά κύτταρα μπορεί να αναπτυχθεί επίσης θυμική και / ή περιφερική ανεκτικότητα. Η αυτό-ανοχή αφορά Τ λεμφοκύτταρα ειδικά για πεπτίδια υψηλής συγγένειας προς το MHC. Αντίθετα, τα Τ κύτταρα που αντιδρούν με όγκους, κατευθύνεται εναντίον πεπτιδίων όγκων με χαμηλή συγγένεια προς το MHC. Το μεγαλύτερο εμπόδιο για τη χρήση τέτοιων πεπτιδίων για ανοσο¬θεραπεία, είναι ότι αυτά είναι ασθενώς ή καθόλου ανοσογόνα. Αυτό οφείλεται στην αδυναμία τους να σχηματίζουν μεγάλο αριθμό σταθερών συμπλοκών MHC/πεπτιδίων στην επιφάνεια των ACP (κύτταρα που παρουσιάζουν αντιγόνα, Antigen Presenting Cells). Στην παρούσα ερ¬γασία περιγράφουμε μία προσέγγιση η οποία επιτρέπει την επαγωγή CTL απόκρισης έναντι χαμηλής συγγένειας HLA Α2.1 περιορισμένων πεπτιδίων. Αυτή συνίσταται στην τροποποίηση της πεπτιδικής αλληλουχίας με την εισαγωγή στη θέση 1 μίας Tyr, η οποία ευνοεί την αλληλεπίδρα¬ση πεπτιδίου/HLA Α2.1. Χρησιμοποιήθηκαν 25 πεπτίδια με διάφορες συγγένειες και ικανότητες σταθεροποίησης με το HLA Α2.1. Η αξιολόγηση της ικανότητα τους να επάγουν CTL απόκριση έδειξε μία αυστηρή συσχέτιση μεταξύ της συγγένειας σύζευξης/ικανότητας σταθεροποίησης και της ανοσογονικότητας. Τα ανάλογά τους με Ρ1Tyr παρουσίασαν υψηλότερη συγγένεια 1,5 έως 55,5 φορές και σταθεροποίησαν το HLA Α2.1 για τουλάχιστον 2 ώρες περισσότερο από τα α¬ντίστοιχα φυσικά πεπτίδια. Επιπλέον, η Ρ1Tyr τροποποίηση δεν μετέβαλε την αντιγονική ειδικό¬τητα των πεπτιδίων, αφού τα ανάλογα με Ρ1Tyr πάντα αναγνωρίζονταν από ειδικά CTL για τα φυσικά πεπτίδια και αντιστρόφως, ειδικά CTL για τα Ρ1Tyr ανάλογα αναγνώρισαν τα φυσικά πεπτίδια. Τελικά, όπως ήταν αναμενόμενο, τα Ρ1Tyr ανάλογα των χαμηλής συγγένειας με το HLA Α2.1 πεπτιδίων νεοπλασιών ενεργοποίησαν αποτελεσματικά in vivo απόκριση ειδικών CTL για τα φυσικά πεπτίδια. / Induction of CTL response to HLA A2.1 restricted tumor peptides exhibiting a low MHC binding affinity and a weak immunogenicity. MHC class I restricted peptides derived from self-proteins overexpressed by a wide variety of human tumor cells are potential targets for a broad spectrum CTL based immunotherapy in cancer. However, since they are presented by normal cells they may also represent thymic and/or peripheral tolerogens. Self-tolerance principally concerns high MHC/affinity peptide specific T cells and the tumor reactive T cells repertoire that is available to be mobilized by peptide based tumor vaccination protocols is directed against low MHC/affinity tumor peptides. A major barrier to the use of low MHC/affinity tumor peptide for immunotherapy is that they are weakly or even not immunogenic. This is due to their inability to form a high number of stable MHC /peptide complexes on the surface of antigen presenting cells. In the present work we describe an approach that allows the induction of a CTL response against low affinity HLA A2.1 restricted tumor peptides. It consisted in modifying the peptide sequence by introducing a Y in the first position known to have a favorable effect in the peptide/HLA A2.1 interaction. Twenty five peptides with variable HLA A2.1 stabilization capacity are included in this study and the evaluation of their capacity to induce a CTL response in a HLA A2.1 transgenic, Db-/-, mb2m-/- mouse showed a strict correlation between MHC binding affinity/MHC stabilization capacity and immunogenicity. Their P1Y variants exhibited a 1.5 to > 55 fold higher affinity and stabilized the HLA A2.1 for at least 2 hrs more than the corresponding native peptides. These effects were more pronounced for the low affinity native peptides. Moreover, P1Y modification did not alter the antigenic specificity of peptides since P1Y variants were always recognized by the native peptide specific CTL and, inversely P1Y specific CTL recognized native peptides. Finally, as expected, P1Y variants of low HLA A2.1 affinity tumor peptides efficiently triggered in vivo a native peptide specific CTL response.

<p>Despite aggressive surgical resection, cytotoxic chemotherapy, and external beam radiotherapy, most cases of glioblastoma (GBM) remain recalcitrant. These outcomes necessitate novel developmental therapeutics that spare normal tissue. Immunotherapy is a promising novel adjuvant treatment that can harness the cytotoxic capacity of the immune system against tumor-associated antigens with exquisite specificity. To circumvent the challenges associated with the advancement of adoptive cellular immunotherapy, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can reshape a host’s immune profile through combinatorial delivery of RNAs encoding for tumor antigens and RNAs encoding for immunomodulatory molecules to mediate long-lived T cell persistence. </p><p>We sought to assess if vaccination with amplified tumor derived RNA encapsulated in lipophilic NPs could be assembled to transfect antigen presenting cells (APCs) in vivo and induce therapeutic anti-tumor immunity in pre-clinical murine tumor models. We hypothesized that RNA encapsulated nanoliposomes would localize to reticuloendothelial organs such as the spleen and liver, transfect APCs therein and induce peripheral antigen specific T cell immunity against GBM. Since activated T cells can cross the blood brain barrier and exert their effector functions against GBM antigens, peripheral transfection of APCs by RNA-NPs represents an attractive vaccination approach for priming endogenous immunity against refractory brain tumors.</p><p>We screened several translatable NP formulations for their ability to transfect dendritic cells (DCs) in vitro with GFP mRNA. We demonstrated that the NP DOTAP was the most promising translatable formulation compared to alternative cationic liposomal preparations and linear polyethylenimine NPs with and without DC targeting mannose receptors. RNA-NP vaccines formulated in DOTAP were shown to induce in vivo gene expression and preserve RNA stability over time. We determined that intravenous (IV) injection of RNA-NPs was requisite for inducing functional antigen specific immunity, which was superior to standard peptide vaccines formulated in complete Freund’s adjuvant (CFA). IV administered RNA-NPs localized to splenic and hepatic white blood cells (WBCs); these cells expanded antigen specific T cells when transferred to naïve immunocompetent mice. RNA-NPs induced increased percentages of B7 co-stimulatory molecules, but also elicited compensatory PD-L1 expression. We enhanced the immunogenicity and anti-tumor efficacy of RNA-NP vaccines by combining RNA-NPs with immune checkpoint blockade against PD-L1. We also enhanced the immunogenicity and efficacy of this platform by simply combining mRNAs encoding for immunomodulatory cytokines (i.e. GM-CSF). Finally, we demonstrated that RNA-NP vaccines mediate anti-tumor efficacy against intracranial and subcutaneous melanomas and engender therapeutic anti-tumor efficacy in a cellular immunotherapy model against a radiation/temozolomide resistant invasive murine high-grade glioma.</p><p>GBM remains invariably associated with poor patient outcomes thus necessitating development of more targeted therapeutics. Clinically translatable RNA-NPs form stable complexes making them amenable to overnight shipping. They induce potent immune responses when administered systemically and mediate robust anti-tumor efficacy that can be enhanced through co-delivery of immunomodulatory RNAs. </p><p>This technology can simultaneously bypass the complexity of cellular therapeutics while cutting down the time to generation of personalized vaccines. Since RNA-NP vaccines can be made within days from a tumor biopsy, providing near immediate immune induction against GBM, these formulations can provide a more feasible and effective therapy with a wide range of applicability for all malignancies that can be targeted using RNA obtained from surgical resection of solid tumors.</p> / Dissertation

Submitted by Wesley Felipe Ferreira Mora Gil null (wes_moragil@hotmail.com) on 2018-04-03T13:14:51Z No. of bitstreams: 1 dissertacao weley versao final.pdf: 3054931 bytes, checksum: 321569a638824c9d0f4d6a48a3d1dee2 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-04-05T13:02:04Z (GMT) No. of bitstreams: 1 gil_wffm_me_bot.pdf: 3054931 bytes, checksum: 321569a638824c9d0f4d6a48a3d1dee2 (MD5) / Made available in DSpace on 2018-04-05T13:02:04Z (GMT). No. of bitstreams: 1 gil_wffm_me_bot.pdf: 3054931 bytes, checksum: 321569a638824c9d0f4d6a48a3d1dee2 (MD5) Previous issue date: 2018-02-16 / Câncer pode ser definido como um crescimento desordenado de células que não permanecem em uma região limitada, invadindo outros tecidos e órgãos. Indicadores mostram que a mortalidade por câncer vem aumentando, por esse motivo é imprescindível a busca por novos tratamentos. A imunoterapia surge como uma modalidade de tratamento promissora, a qual utiliza-se do sistema imunológico no combate ao câncer. Outra tendência na oncologia é a combinação de diferentes modalidades de tratamentos. Neste trabalho, propomos um modelo matemático de equações diferenciais ordinárias, com o intuito de analisar como o tratamento imunoterápico e quimioterápico podem auxiliar um ao outro no tratamento do câncer. Utilizamos um software matemático para a construção dos retratos de fase e o método Runge-Kutta de quarta ordem para as simulações numéricas. As simulações mostraram que a imunoterapia e a quimioterapia podem levar à eliminação das células e uma sobrevida maior após o tratamento. É exibido também que citotoxicidade da quimioterapia é fundamental para o sucesso do tratamento. / Cancer may be defined as a uncontrolled growth of cells that do not remain in a limited region, invading other tissues and organs. Indicators show that mortality from cancer is increasing, so the search for new treatments is essential. Immunotherapy appears as a promising treatment modality, which uses the immunological system in the fight against cancer. Another trend in oncology is the combination of different treatment modalities. In this work, we propose a mathematical model of ordinary differential equations, in order to analyze how immunotherapeutic and chemotherapeutic treatment can help one another reciprocally. We use a mathematical software for the construction of the phase portraits and the method fourth-order Runge-Kutta for numerical simulations. The simulations have shown indications that immunotherapy may assist the chemotherapy by causing cure or by allowing a longer overlife after treatment. It is also shown that cytotoxicity of chemotherapy is critical to successful treatment.

Orientador: Paulo Fernando de Arruda Mancera / Resumo: Câncer pode ser definido como um crescimento desordenado de células que não permanecem em uma região limitada, invadindo outros tecidos e órgãos. Indicadores mostram que a mortalidade por câncer vem aumentando, por esse motivo é imprescindível a busca por novos tratamentos. A imunoterapia surge como uma modalidade de tratamento promissora, a qual utiliza-se do sistema imunológico no combate ao câncer. Outra tendência na oncologia é a combinação de diferentes modalidades de tratamentos. Neste trabalho, propomos um modelo matemático de equações diferenciais ordinárias, com o intuito de analisar como o tratamento imunoterápico e quimioterápico podem auxiliar um ao outro no tratamento do câncer. Utilizamos um software matemático para a construção dos retratos de fase e o método Runge-Kutta de quarta ordem para as simulações numéricas. As simulações mostraram que a imunoterapia e a quimioterapia podem levar à eliminação das células e uma sobrevida maior após o tratamento. É exibido também que citotoxicidade da quimioterapia é fundamental para o sucesso do tratamento. / Abstract: Cancer may be defined as a uncontrolled growth of cells that do not remain in a limited region, invading other tissues and organs. Indicators show that mortality from cancer is increasing, so the search for new treatments is essential. Immunotherapy appears as a promising treatment modality, which uses the immunological system in the fight against cancer. Another trend in oncology is the combination of different treatment modalities. In this work, we propose a mathematical model of ordinary differential equations, in order to analyze how immunotherapeutic and chemotherapeutic treatment can help one another reciprocally. We use a mathematical software for the construction of the phase portraits and the method fourth-order Runge-Kutta for numerical simulations. The simulations have shown indications that immunotherapy may assist the chemotherapy by causing cure or by allowing a longer overlife after treatment. It is also shown that cytotoxicity of chemotherapy is critical to successful treatment. / Mestre

Meduloblastoma (MB) e Tumor Teratóide Rabdóide Atipico (TTRA) são tumores malignos do sistema nervoso central (SNC) que ocorrem na infância. Embora tenha havido um aumento na sobrevida, recorrência e metástases são frequentes e as opções terapêuticas poucas e tóxicas para crianças. Uma alternativa à terapia tradicional é a imunoterapia, que pode contornar os efeitos tóxicos associados à radioterapia e quimioterapia. De uma forma geral a imunoterapia depende da presença de antígenos associados a tumor (TAA) e/ou processamento de antígenos expressos pelo HLA classe I. No entanto, TAA para MB ou ATRT não estão bem definidos e tecidos neuronais tem a expressão de HLA classe I muito baixa. As células Natural Killer (NK) não dependem de TAA para citólise e são particularmente ativas na ausência de moléculas do HLA classe I tendo, portanto, potencial para o tratamento dessas doenças. Atualmente as barreiras para a aplicação clínica de células NK são quantitativas e qualitativas. Com o intuito de quebrar a barreira quantitativa foi criada uma plataforma tecnológica para a expansão das NK ex vivo. Essa plataforma consiste no co-cultivo de células mononucleares de sangue periférico com células apresentadoras de antígeno artificiais (aAPC), que expressam Interleucina 21 ligada à membrana. Com essa tecnologia foi possível expandir células NK na ordem de 40.000 vezes. Também foi demonstrada a persistência das células NK expandidas in vitro por 3 semanas após infusão em cérebro murino e que essas células expressam altos níveis de citocinas antitumorais e estimulantes do sistema imune: Interferon gama e Fator de Necrose Tumoral alfa. Finalmente, foi demonstrada a atividade citolítica das células NK tanto in vitro, contra um painel de células de MB e ATRT, quanto in vivo em um modelo murino de MB. Os resultados obtidos fornecem a primeira evidência pré-clínica que provê suporte ao uso de células NK expandidas usando essa plataforma tecnológica no combate a cânceres de cérebro pediátricos. Com base nesses dados, nosso grupo no MD Anderson Cancer Center iniciou um ensaio clínico fase I para testar a segurança e eficácia de administração locorregional de células NK expandidas usando aAPC para o tratamento de tumores pediátricos da fossa posterior do cérebro. / Medulloblastoma (MBs) and Atypical Teratoid/Rhabdoid Tumor (ATRT) are malignant pediatric brain tumors. Although survival has improved, recurrence and metastasis are frequent with few therapeutic options for these children. Immunotherapy is an alternative to traditional therapies that may circumvent the potential toxicities associated with traditional chemotherapy and radiation approaches. Many immune based therapies rely on the presence of tumor associated antigens (TAAs) and/or antigen processing and class I human leukocyte antigen (HLA I) expression. However, TAAs for MB are not well defined and neuronal tissues have very low HLA I expression. Natural killer (NK) cells do not rely on TAA for cytolysis and therefore have potential for the treatment of these diseases. The current barriers to clinical application of NK therapy are quantitative and qualitative. To overcome quantitative barriers, we have worked with a platform technology for the ex vivo expansion of NK cells through co-culture of peripheral blood mononuclear cells with artificial antigen presenting cells expressing membrane-bound IL-21 (mbIL21) to promote a 40,000-fold expansion of NK cells. We also demonstrate prolonged life-span of ex vivo expanded NK cells and persistence for up to 3 weeks post-infusion in the murine brain. These cells express high levels of immune stimulatory and anti-tumor cytokines - interferon gamma and TNF-alpha following activation. Finally we demonstrate NK cytolytic activity in vitro against a panel of primary and established ATRT and MB cells and in vivo following locoregional delivery in a mouse orthotopic model of MB. Our data provide the first pre-clinical evidence supporting the use of mbIL21 expanded NK cells against pediatric brain tumors. Based on this data we have initiated a novel Phase I clinical trial at MD Anderson Cancer Center to assess the safety and efficacy of locoregional delivery of mbIL21 expanded NK cells for the treatment of posterior fossa pediatric brain tumors.

Lung cancer is the leading cause of cancer mortality worldwide. Understanding biological processes of specific antitumor immune response remains of an eminent interest and represents necessity for designing successful antitumor immunotherapeutic strategies. The theoretical part of the thesis describes components of the immune system that are involved in antitumor response and discusses their role in the hitherto known and used lung cancer immunotherapy. In the practical part of the thesis, two studies studying different aspects of anticancer immune response are described. Both studies were conducted in cooperation with 3rd Surgical Department 1st Faculty of Medicine, Charles University and University Hospital Motol and with the biotechnology company Sotio a.s. The first study is focused on the humoral component of the specific antitumor response and prospectively analyses serum frequency of antitumor antibodies against NY-ESO-1, Her2/neu and MAGE-A4 antigens in 121 patients with NSCLC. Here it was shown for the first time that tobacco smoking significantly increases the frequency of NY- ESO-1 antibodies in sera of smokers in comparison to ex-smokers and non-smokers. The second study is focused on the cellular component of the specific antitumor response investigating the activity of the dendritic...

Liver metastases are the most common cause of death in colorectal cancer patients. The standard of care and potential for cure for colorectal liver metastases is resection, but often times disease it too extensive for this treatment. Over the years, cancer research has made way for advances in treating progressive disease through immunotherapy. By genetically modifying an individual’s immune system using virally transduced chimeric antigen receptor T cells (CAR-T), patients are better able to receive exquisitely specific T cells to target specific tumors. Furthermore, selective delivery strategies may enhance efficacy while limiting detrimental, systemic adverse effects. Not only this, CAR-Ts have also lead to complete remission in some liquid tumors while maintaining the potential for remission in solid tumors as well. This literature review takes readers through the emergence of the different generations of CAR-T and the various studies including clinical trials that have demonstrated the safety and efficacy of CAR-T. The second portion of this paper will outline the design for a phase II clinical trial using intrahepatic CAR-T therapy in addition to selective internal radiation therapy (SIRT) for refractory CEA+ colorectal liver metastases. Benefits and limitations of using these therapies are further discussed.

In the first part of the theses, I studied therapeutical effect of intratumoral application of various types of bacteria on melanoma B16-F10 bearing mice alone or in combination with another immunostimulatory compounds. Tumour size, metastasis and survival were monitored. The second part was focused on study of cytotoxic effect of neutrophils on melanoma cells and the role of neutrophil extracellular traps formation.

The aim of thesis was to study murine melanoma B16- F10 therapy based on the use of TLR agonists combinedwith activation of phagocytosis. Mechanisms of this therapy were studied on the bases of analysis of tumor infiltrating immune cells and evaluationof thein effect on tumor cells.

Infusões de células T e células NK (Natural Killer) de sangue periférico estão sendo realizadas para tratamento de malignidades. Os linfócitos propagados ex vivo, em normóxia (20% O2), são intravenosamente infundidos e precisam sobreviver a hipóxia associada a circulação venosa, da medula óssea (5% O2) e do microambiente tumoral (1% O2). O objetivo principal deste estudo foi determinar a capacidade proliferativa das células humanas T e NK em normóxia (20% O2) versus hipóxia (1% O2), por 28 dias, utilizando uma célula apresentadora de antígeno artificial (aAPC) para propagação em grau clínico. As células T expostas a hipóxia cresceram 100 vezes menos que as células T cultivadas em normóxia, enquanto que houve uma diminuição de 1000 vezes na taxa proliferativa das células NK hipóxicas, que exibiram um aumento na apoptose bem como um prejuízo na citotoxicidade. Hipóxia também induziu uma diminuição na expressão dos receptores KIR, NCR e NKG2D das células NK. Nesta mesma condição, a produção de IL-2 e IFNγ nas células T estavam diminuídas, sendo que nas células NK esse efeito foi mais acentuado. Hipóxia aumentou a expressão de genes relacionados com apoptose, angiogênese e metabolismo glicolítico, os quais estavam moderadamente aumentados nas células T, mas profundamente super-regulados nas células NK. Os níveis de ATP nas células T foram muito similares em ambas as condições de oxigênio, mas intensamente diminuídos nas células NK cultivadas em hipóxia. Também se observou, que a expressão do miR-210 induzido por hipóxia, estava super regulada nas células NK hipóxicas correlacionando com a perda da expressão da molécula NCAM/CD56. Em conjunto, os resultados deste estudo demonstram um maior impacto da hipóxia sobre as atividades proliferativas e citotóxicas das células NK estimuladas por aAPCs. Estudos adicionais são necessários para o entendimento do impacto deste comportamento das células NK em condições hipóxicas sobre a imunoterapia celular adotiva. / Infusions of T cells and natural killer (NK) cells from peripheral blood (PB) are being undertaken for the treatment of malignancies. Lymphocytes are propagated ex vivo in normoxia (20% O2) and intravenously infused and must survive hypoxia associated with venous blood and bone marrow (5% O2), and the tumor environment (1% O2). The objective this study was to determine the ability of T and NK cells to proliferate under normoxia (20% O2) versus hypoxia (1% O2) over 28 days using an artificial antigen presenting cells (aAPC) to propagate clinical-grade lymphocytes. T cells continuously exposed to 4 weeks of hypoxia grew at a rate of 100-fold less than T cells cultured in normoxia while the proliferative rate of NK cells lagged by 1,000-fold, behind normoxic conditions. Hypoxic cultured NK cells exhibit an increase in apoptosis as well as a correspondent impairment in cytotoxicity. In low oxygen tension the expression of KIR, NCR, and NKG2D receptors were decreased in NK cells. In hypoxia, the production of IL-2 and IFNγ were decreased in T cell and more so in NK cell. Chronic hypoxia increased the expression of related apoptosis, glycolytic metabolism and angiogenesis genes which were moderately increased in T cells but profoundly upregulated in NK cells. ATP levels in T cell were very similar in both oxygen conditions, but profoundly diminished in NK cells under hypoxia. We also noted that hypoxia inducible miR-210 levels are up regulated in hypoxic NK cells correlating with loss of CD56 expression. Taken together, this data show that a greater impact of hypoxia on proliferative and cytotoxic activity of NK cells activated by artificial antigen-presenting cells. More studies are needed to understand the impact of this behavior on the cell adoptive immunotherapy.