Immunotoxins (ITs), which consist of antibodies conjugated to toxins, have been proposed as a treatment for cancer and chronic infections. To develop and improve the ITs, different toxins such as ricin, have been used, aiming for higher efficacy against target cells. The toxin pulchellin, isolated from the Abrus pulchellus plant, has similar structure and function as ricin. Here we have compared two plant toxins, recombinant A chains from ricin (RAC) and pulchellin (PAC) toxins, for their ability to kill HIV Env-expressing cells. Briefly, RAC and PAC were produced in E. coli, and chromatographically purified, then chemically conjugated to two different anti-HIV monoclonal antibodies (MAbs), anti-gp120 MAb 924 or anti-gp41 MAb 7B2. These conjugates were characterized biochemically by microcapillary electrophoresis and BCA assay and immunologically by a variety of ELISA tests. We performed a side-by-side comparison of their ability to bind, enter and kill HIV infected cells (H9/NL4-3) or Env-transfected 293T cells, as well as their non-specific toxicity on uninfected or non-transfected parental cells. Cell binding and internalization were studied by flow cytometry and confocal microscopy. Results showed that PAC can function within an effective IT. The ITs demonstrated specific binding against native antigens on persistently HIV-infected cells and recombinant antigens on Env-transfected cells. An irrelevant antibody conjugated to either RAC or PAC had no effect. PAC cytotoxicity appears somewhat less than RAC, the standard for comparison. This is the first report that PAC may have utility for the design and construction of therapeutic ITs, highlighting the potential role for specific cell targeting not only for AIDS also for cancer therapy. / As toxinas imunológicas (TIs), que consistem em anticorpos conjugados com toxinas, foram propostas como tratamento para câncer e infecções crônicas. Para desenvolver e melhorar as TI, diferentes toxinas, como a ricina, foram usadas, visando uma maior eficácia contra células alvo. A toxina pulchellina, isolada da planta de Abrus Pulchellus, tem estrutura e função semelhantes à da ricina. Aqui, comparamos duas toxinas de plantas, cadeias A recombinantes de toxinas de ricina (RAC) e pulchellina (PAC), por sua capacidade de matar células que expressam HIV. Resumidamente, RAC e PAC foram produzidos em E. coli e purificados por cromatografia, depois conjugados quimicamente com dois anticorpos monoclonais anti corpo-HIV diferentes (MAcs), MAc 924 anti-gp120 ou MAc 7B2 anti-gp41. Estes conjugados foram caracterizados bioquimicamente por eletroforese microcapilar e teste BCA e imunologicamente por uma variedade de testes ELISA. Realizamos uma comparação lado-a-lado de sua capacidade de ligar, entrar e matar células infectadas pelo HIV (H9 / NL4-3) ou células 293T transfectadas com Env, bem como a sua toxicidade não específica em parentes não infectados ou não transfectados Células. A ligação celular e a internalização foram estudadas por citometria de fluxo e microscopia confocal. Os resultados mostraram que PAC pode funcionar dentro de uma TI efetiva. As TI demonstraram ligação específica contra antígenos nativos em células persistentemente infectadas pelo HIV e antígenos recombinantes em células transfectadas com Env. Um anticorpo irrelevante conjugado com RAC ou PAC não teve efeito. A citotoxicidade de PAC aparece um pouco menor que o RAC, o padrão de comparação. Este é o primeiro relatório que o PAC pode ter utilidade para o projeto e a construção de TI terapêuticas, destacando o papel potencial para o direcionamento celular específico, não apenas para a AIDS, também para a terapia do câncer.

Esporotricose é uma micose subcutânea, causada principalmente pelo fungo Sporothrix schenckii e a nova espécie Sporothrix brasiliensis. Foi relatado na literatura que os camundongos infectados com a cepa de S. schenckii M-64 produziu uma resposta imune mista, em que o soro dos camundongos infectados reagiram apenas com uma glicoproteína de 70 kDa (gp70), identificada como importante fator de virulência. Assim o presente trabalho visa avaliar a importância à virulência e eficácia do anticorpo monoclonal P6E7 em outras cepas do complexo Sporothrix. Camundongos foram infectados com as cepas de S. schenckii (1099-18 e 15383) e S. brasiliensis (5110 e 17943). Cada 7 dias, o baço e fígado foram retirados para a análise do UFC e citocinas. Foi realizado western blot com o soro dos camundongos infectados e com anticorpo monoclonal P6E7 utilizando o exoantígenos das diferentes cepas. Foi realizado protocolo de tratamento com o anticorpo P6E7 nos camundongos infectados, para a avaliação da carga fúngica no baço e no fígado. Análise de citocinas mostrou que as cepas de S. schenckii induziram uma resposta mista Th1/Th2, entretanto nos camundongos infectados com as cepas de S. brasiliensis, não foi observada produção significativa de citocinas. No western blot realizado com exoantígenos das cepas de Sporothrix sp. foi observado diferentes componentes antigênicos, quando o soro de camundongos infectados foi utilizado, porém a reação com anticorpo monoclonal contra a gp70 demonstrou que a gp70 estava presente apenas no exoantígeno da cepa mais virulenta 5110. Infecção realizada com todas as cepas demonstrou cronicidade pela análise UFC. O tratamento com o AcMo P6E7 foi eficaz no tratamento da esporotricose experimental, sendo capaz de diminuir a carga fúngica, principalmente no baço dos camundongos infectados. / Sporotrichosis is a subcutaneous mycosis, caused mainly by fungi Sporothrix schenckii and the new specie Sporothrix brasiliensis. Was reported in previous studies that mice infected with low virulent S. schenckii M-64 strain, produced a mixed immune response, where the infected mice serum reacted only with a 70 kDa glycoprotein (gp70), identified as important virulence factor. Thus, the present study aims to evaluate the virulence importance and effectiveness of monoclonal antibody P6E7 in other Sporothrix complex strains. Mice were infected with Sporothrix yeast strain of S. schenckii (1099-18 and 15383) and S. brasiliensis (5110 and 17943). Each 7 days, the spleen and liver were taken for CFU and cytokines analysis. The western blot was performed with the serum obtained from infected mice and a monoclonal antibody P6E7 using exoantigens from different strains. Was performed treatment protocol with the monoclonal antibody P6E7 on infected mice, for spleen and liver fungal burden evaluation. Cytokine analysis showed that S. schenckii strains induce a mixed Th1/Th2 response, however in the S. brasiliensis strains wasn\'t observed significant cytokine production. In western blot accomplished with the strains exoantigens was observed different antigenic components when the infected mice serum was used, however with monoclonal antibody against gp70 demonstrated that gp70 was present only in most virulent strain 5110. Infection performed with all strains demonstrated chronicity for CFU analysis. Treatment with Mab P6E7 was effective in experimental sporotrichosis, with reducing capable of fungal load, mainly in the spleen of infected mice.

Les anticorps monoclonaux (AcM) sont désormais considérés comme une alternative thérapeutique crédible pour traiter les infections virales graves. Comprendre leurs multiples mécanismes d’action est donc crucial pour améliorer leur effet thérapeutique. En utilisant un modèle d’infection virale chez la souris (leucémie induite par le rétrovirus FrCasE), l’équipe a montré qu’une immunothérapie courte par un AcM neutralisant induisait une immunité antivirale protectrice sur le long-terme (effets « vaccinaux ») qui est dépendante du fragment Fc de l’AcM. Ainsi, des immuns complexes (IC) formés à partir de l’AcM thérapeutique et de déterminants viraux, induisent l’activation de cellules immunitaires, notamment les cellules dendritiques (DCs), via leur interaction avec les FcRs exprimés à la surface des cellules. Cependant, ces interactions IC-FcR peuvent également concerner d’autres cellules du système immunitaire outre que les DCs, telles que les macrophages, monocytes ou bien encore les neutrophiles, qui expriment elles aussi les FcRs à leur surface et ce de façon différentielle. Dans ce contexte, il est important d’identifier quels FcRs et quelles cellules les exprimant sont essentiels à l’induction des effets vaccinaux par les AcM. C’est pourquoi mes travaux thèse se sont focalisés sur l’étude du rôle des neutrophiles et des FcγRs dans la modulation de la réponse immune par les AcM. Cette étude repose sur le caractère Fc-dépendant de l’induction d’une réponse immune protectrice par les AcM ainsi que sur les propriétés immunomodulatrices des neutrophiles, qui ont été décrites dans différents contextes pathologiques mais jamais étudiées dans le cadre d’une immunothérapie antivirale par AcM. Pour cela, j’ai utilisé différentes approches in vitro, ex vivo et in vivo.En utilisant le modèle d’infection par FrCasE, il a été montré que les neutrophiles ainsi que le FcγRIV ont un rôle crucial dans l’induction des effets vaccinaux par les AcM, notamment via l’induction d’une réponse humorale antivirale endogène protectrice à très long-terme. De plus lors d’expériences in vitro, il a également été souligné que les neutrophiles sont plus efficacement activés par les IC comparé au virus seul et que différentes cytokines pro-inflammatoires et/ou immunomodulatrices (telles que le TNF et les intérferons de type I et II) potentialisent l’activation des neutrophiles induite par les IC. Mes travaux ont aussi mis en évidence que l’infection virale et l’immunothérapie modulent l’expression des FcRs, et notamment induisent la surexpression du FcRIV sur deux populations distinctes de neutrophiles (différentiées par le niveau d’expression du marqueur de surface Ly6G: Ly6Ghi et Ly6Gint) et sur les monocytes inflammatoires. Enfin, mes travaux montrent que l’immunothérapie par AcM module les profils de sécrétion chimiokinique et cytokinique de ces 3 types cellulaires surexprimant le FcRIV, bien que la nature des profils de sécrétion varie en fonction du type cellulaire et évolue au cours du temps. Ces résultats suggèrent que l’effet immunomodulateur des AcM repose sur l’activation de différents acteurs de la réponse immunitaire précoce, en induisant la sécrétion de chimiokines et de cytokines nécessaires à l’orchestration de la réponse immune. Ils suggèrent aussi une coopération entre ces différents acteurs dans la mise en place d’une immunité protectrice.Pour finir l’ensemble de mes travaux ont mis en évidence un rôle immunomodulateur clé du FcyRIV, ainsi que des différentes cellules l’exprimant, dans l’induction d’une réponse immune protectrice induite par des AcM antiviraux. Ces révélations pourraient avoir des conséquences importantes dans l'amélioration des immunothérapies à base d'AcM. / Monoclonal antibodies (mAbs) are now considered as a true therapeutic alternative for treating severe viral infections. Figure out their multiple mechanisms of action is therefore crucial to improve their therapeutic effect. Using a mouse model of viral infection (the FrCasE retrovirus-induced leukemia), the team showed that a short immunotherapy with a neutralizing mAb induces long-term protective antiviral immunity ("vaccine" effects) which is Fc-dependent. Notably, immune complexes (IC) formed with therapeutic mAbs and viral determinants induce the activation of immune cells, especially dendritic cells (DCs) via their interaction with FcγRs expressed on the cell’s surface. However, IC-FcγR interactions can involve different cells of the immune system in addition to DCs, such as macrophages, monocytes or neutrophils, which differentially express FcγRs. In this context, it is important to identify which FcγRs and which FcγR-expressing cells are crucial in the induction of vaccine effects induced by mAbs. It’s the reason why my thesis work has focused on the study of the role of neutrophils and FcγRs in the modulation of immune response by mAbs. This study is based on the Fc-dependent nature of the induction of a protective immune response by mAbs and the immunomodulatory properties of neutrophils, described in different pathological situations but never studied in an mAbs antiviral immunotherapy context. To this end, I used different approaches in vitro, ex vivo and in vivo.By using the FrCasE infection model, it has been shown that neutrophils as well as FcγRIV have a crucial role in the induction of vaccine effects by mAbs, notably via the induction of a long-term protective antiviral humoral response. Moreover the in vitro experiments, highlighted that neutrophils are more effectively activated by IC compared to virus alone and that different pro-inflammatory and/or immunomodulating cytokines (i.e.TNFα and type I and type II interferons) potentiate the activation of neutrophils induced by IC. My work also revealed that viral infection and immunotherapy modulate the expression of different FcγRs, and notably they induce the overexpression of FcγRIV on two distinct populations of neutrophils (differentiated by their expression levels of the Ly6G surface marker: Ly6Ghi and Ly6Gint) and inflammatory monocytes. Finally, my work shows that immunotherapy with Mab modulates the chemokinic and cytokinic secretion profiles of these 3 FcγRIV-over-expressing cell, although the nature of the secretion profiles differs according to the cell type and evolves over time. These results suggest that the immunomodulatory effect of mAbs is based on the activation of different actors of the early immune response by inducing the secretion of chemokines and cytokines necessary for the orchestration of the immune response. They also suggest a potential cooperation between these different actors in the establishment of protective immunity.Altogether, these results show a key immunomodulator role of FcγRIV as well as of different cells expressing it in the induction of a protective immune response by antiviral mAb. They might have important consequences for the improvement of Mab-based immunotherapies.

This thesis describes the synthesis and radiohalogenation of compounds of potential use for tumor targeting. The first section describes the synthesis and radioiodination of DNA intercalating compounds. The compounds are derivatives of 9-aminoacridine, and the anthracyclins daunorubicin and doxorubicin. The precursor compounds were labeled with 125I (T1/2 = 60 days), which is an Auger emitting nuclide. 125I decaying in the close vicinity of DNA is known to have a much higher cell killing effect than 125I decaying in the cytoplasm and some of the labeled compounds prepared in this thesis are currently being tested for use in targeted radionuclide therapy for cancer. The second section describes the radiobromination of closo-carboranes by subjecting the corresponding iodinated compounds to palladium-catalyzed halogen exchange using [76Br]bromide. The 76Br isotope (T1/2 = 16.2 h) is a positron emitting nuclide that is suitable for PET studies. Via the halogen exchange reaction good to excellent radiochemical yields of radiobrominated closo-carboranes were obtained. The results of the present study may prove to be applicable to pharmacokinetic studies of carboranes and their derivatives. The third and final section describes the indirect radiobromination of the trastuzumab anti-HER2 monoclonal antibody and of the anti-HER2 Affibody by means of an “one-pot” procedure using N-succinimidyl-5-(tributylstannyl)-3-pyridinecarboxylate (SPC) and ((4-hydroxyphenyl)ethyl))maleimide (HPEM), respectively. It was found that SPC and HPEM can be efficiently radiobrominated and thereafter coupled to the antibody and Affibody, respectively. The labeled proteins retained their capacity to bind specifically to HER2 expressing SKOV-3 cells in vitro. Application of this method to 76Br might enable the use of PET in the detection of HER2 expression in breast, ovarian, and urinary bladder carcinomas.

Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral disease in the world and therefore is considered an emerging global health threat. The four DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that infect humans are distinguished from one another by unique antigenic determinants (epitopes) on the DENV envelope (E) protein. The E protein is the primary antigenic site of the DENV and is responsible for inducing neutralising antibody (Ab) and cell mediated immune response in DENV infected hosts. The DENV E protein also mediates attachment of virions to host cell receptors and entry of virions into host cells by membrane fusion. The study of epitopes on DENV E protein is necessary for understanding viral function and for the design of unique polyvalent vaccines capable of inducing a neutralising antibody response against each DENV serotype. Reverse genetics using infectious cDNA clones has enabled the construction of functional intertypic DENV, where the E protein of one DENV serotype is put in the genetic background of a different DENV serotype. In addition, observations from our laboratory indicate that chimeric E proteins, consisting of E protein structural domains from different DENV serotypes can fold into functional proteins. This suggests that there is potential to engineer viruses with intertypic DENV E proteins as potential DENV vaccine candidates, which is the long term goal of studies within our research group. However, if a chimeric E protein was to be constructed containing epitopes involved in antibody mediated neutralisation of each DENV serotype, then knowledge of the location of these epitopes on the E protein of each DENV serotype would be essential. Prior to this study, monoclonal antibodies (MAbs) had been used to identify epitopes involved in antibody mediated neutralisation on the E protein of all DENV serotypes, except DENV-4. The primary objective of this study was to identify epitopes on the DENV-4 E protein involved in neutralisation by antibodies. In order to achieve this objective, a panel of 14 MAbs was generated against DENV-4 in BALB/c mice and characterised using various serological and functional assays. The identification of DENV-4 specific neutralising MAbs in the panel was essential for subsequent experiments aimed at determining antigenic domains, structural domains or specific epitopes (peptides or amino acids) involved in the neutralisation of DENV-4. The majority of MAbs (11/14) generated against DENV-4 recognised the E protein. The remaining three MAbs reacted with the non-structural (NS) 1 protein. The majority of MAbs against the E protein were DENV or Flavivirus group reactive, but four MAbs were DENV-4 specific. All MAbs against the E protein recognised conformationally dependent epitopes and were able to capture DENV-4 in an enzyme linked immuno-adsorbent assay (ELISA). Eighty percent (9/11) of the anti-E MAbs produced for this study neutralised infection of cells by DENV-4 in vitro. Three of the neutralising MAbs (F1G2, 18F5 and 13H8) were DENV-4 specific and also demonstrated the strongest neutralisation activity of the panel, reducing DENV-4 infectivity by 100-1000 fold. The amount of virus neutralised by the MAbs was not related to the avidity of the MAbs. The DENV-4 specific MAbs F1G2, 18F5 and 13H8 were used to identify epitopes involved in neutralisation of DENV-4. The MAbs that effectively captured DENV-4 were used in competitive binding assays (CBAs) to determine spatial relationships between epitopes and therefore define antigenic domains on the DENV-4 E protein. The CBAs indicated that the epitopes recognised by the panel of MAbs segregated into two distinct domains (D4E1 and D4E2) and both contained epitopes involved in neutralisation. CBAs incorporating human serum from DENV-4 infected patients suggested that the MAbs recognised the same, or spatially related, epitopes in domain D4E2 as antibodies from humans who had experienced natural dengue infections, indicating the clinical relevance of such epitopes for the development of DENV vaccines. The reactivity of the capture MAbs with low pH treated DENV-4 was also evaluated in an attempt to identify epitopes that might be more accessible during low pH-mediated virus fusion. Only one of the MAbs (13H8) recognised an acid resistant epitope. Initial attempts to identify epitopes on the DENV-4 E protein involved in neutralisation followed the traditional epitope mapping approach of selecting subpopulations of DENV-4 which escaped neutralisation by MAbs. These attempts were unsuccessful so a variety of strategies for mapping epitopes were used including DENV-4 variant analysis and site directed mutagenesis of the DENV-4 E protein, MAb screening of chimeric DENV-3/4 E proteins and MAb screening of a bacterial peptide display library. DENV-4 variants including DENV-4 isolates from different geographical locations or chemically mutagenised DENV-4 were screened with neutralising MAbs to identify neutralisation escape mutant (n.e.m.) viruses. Site directed mutagenesis of the DENV-4 E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were essential for the binding of neutralising MAbs to an epitope. The MAb screening of DENV-4 variants identified n.e.m.s with amino acid changes at residues E95, E96, E156, E157, E203, E329 and E402 of the DENV-4 E protein. Site directed mutagenesis of the DENV-4 E protein identified two epitopes recognised by the DENV-4 specific neutralising MAbs F1G2 and 18F5 at specific amino acid residues within domains II and III of the DENV-4 E protein. No specific epitopes were identified for the MAb 13H8; however this MAb did recognise domain I and II of the DENV-4 E protein, when screened against DENV-3/4 chimeric DENV E proteins. The first epitope, which was recognised by the MAb F1G2, contained residue E95 which was located in domain II of the DENV-4 E protein. The aspartate (Asp) to alanine (Ala) change at E95 prevented the binding of F1G2 to the DENV-4 E protein. The binding of F1G2 to the E95 residue was confirmed using the pFlitrX bacterial peptide display library, which demonstrated binding of F1G2 to a peptide homologous with residues E99-E104. No peptides recognised by 13H8 and 18F5 were identified by this method. The MAb F1G2 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric DENV E proteins. This implied that F1G2 may be recognising a discontinuous epitope consisting of domains II and III. The second epitope, which was recognised by MAb 18F5, contained residue E329 which was located in domain III of the DENV-4 E protein. The alanine (Ala) to threonine (Thr) change at E329 prevented the binding of 18F5 to the DENV-4 E protein. MAb 18F5 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric E proteins, thus confirming the E329 epitope. The potential mechanisms by which the DENV-4 specific MAbs neutralise virus infection were evaluated by the virus overlay protein binding assay (VOPBA). The binding of MAb 18F5 to a domain III (E329) epitope of the DENV-4 E protein and the binding of MAb F1G2 to domain II (E95, E99-E104) and domain III epitopes (chimeric E protein) of the DENV-4 E protein, prevented the attachment of DENV-4 to a 40 kDa C6/36 cell protein. In contrast the binding of MAb 13H8 to domains I and II of the DENV-4 E protein did not prevent attachment of DENV-4 to the same protein. This was preliminary evidence that the binding of domain III epitopes by the MAbs F1G2 and 18F5 may be important in preventing virus attachment. The binding of MAb 13H8 to domains I and II, and the ability of this MAb to recognise DENV-4 treated at low pH, suggested that MAb 13H8 may block epitopes exposed at low pH that are required for low pH mediated virus fusion to host cell membranes. Overall, the different methods used in this study identified epitopes involved in the neutralisation of DENV-4. The distribution of epitopes involved in neutralisation throughout the DENV-4 E protein were similar to the distribution of epitopes involved in neutralisation on the DENV-1, 2 and 3 E proteins. This suggested that it might be possible to elicit neutralising antibodies against multiple DENV serotypes using chimeric E-proteins derived from two or more DENV serotypes and therefore, facilitate the design of novel tetravalent DENV vaccines.

Le virus de l’hépatite C (VHC) est une des causes majeures de carcinome hépatocellulaire (CHC), dont les traitements ont une efficacité modéré. La transplantation hépatique (TH) est l’option thérapeutique de choix mais est limitée, chez les patients chroniquement infectés par le VHC, par une réinfection systématique du greffon. Nous proposons d’utiliser des lymphocytes génétiquement modifiés (LGM) issus de donneurs sains qui ont fait leurs preuves pour le traitement d’hémopathies malignes et qui,exprimant un gène suicide, peuvent être éliminés en cas d’effets secondaires. Nous montrons maintenant que ces LGM présentent d’une part une activité anti-tumorale vis-à-vis du CHC, et d’autre part un effet anti-viral envers le VHC. L’objectif est la création d’une banque de LGM allogéniques prêts-à-l’emploi, avec des avantages en termes de coût, de logistique et de disponibilité immédiate par rapport aux approches classiques d’immunothérapies, autologues pour la plupart. Parallèlement, nous avons étudié in vivo l’effet anti-viral d’un anticorps monoclonal anti-claudin-1 dirigé contre un co-récepteur duVHC et inhibant l’entrée du VHC dans les hépatocytes humains. Grâce à un modèle d’infection par VHC de souris présentant un foie chimérique humanisé, nous montrons que cet anticorps permet de prévenir efficacement l’infection par le VHC. Nos résultats apportent les preuves de concept de l’utilisation de deux produits, anticorps anti-claudin-1 et LGM, pour la prévention de la réinfection du greffon hépatique par le VHC, les LGM pouvant également être envisagés en association avec les thérapies actuelles du CHC. / The hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC),whose treatments are of limited efficacy. The liver transplantation (LT) is the optimal therapy but is limited by a very rapid and universal HCV reinfection of the liver graft. We propose to use healthy donor-derived suicide gene modified lymphocytes (GML), known to be efficient for the treatment of hematological malignancies and that can be eliminated in case of adverse events. We show now that GML have a strong anti-tumoral activity against HCC and an anti-viral effect on HCV. Our objective is to create a bank of ready for-use allogeneic GML which could have numerous advantages in terms of costs,logistics and immediate availability, as compared with autologous immunotherapies. In parallel, we have studied the in vivo anti-viral effect of a monoclonal antibody (mAb) directed against an HCV coreceptor, claudin-1, inhibiting HCV entry in human hepatocytes. Using a human liver-chimeric mouse model of HCV infection, we show that this mAb can efficiently prevent HCV infection in vivo. Our results provide the proofs of concept that these two products, anti-claudin-1 mAb and GML, could be used for the prevention of liver graft HCV reinfection, while GML could further be used for the treatment of HCC, in combination with current HCC therapies

Linfomas são cânceres que se iniciam a partir da transformação maligna de um linfócito no sistema linfático. Os linfomas são divididos em duas categorias principais: os linfomas de Hodgkin e todos os outros linfomas, denominados linfomas não-Hodgkin (LNH). Os pacientes com LNH são comumente tratados com radioterapia apenas ou combinada com quimioterapia utilizando-se de anticorpo monoclonal anti-CD20, principalmente o rituximab (MabThera&reg). O uso de anticorpos monoclonais (Acm) conjugados à quelantes bifuncionais radiomarcados com radionuclídeos metálicos ou lantanídeos é uma realidade de tratamento para portadores de LNH pelo princípio de radioimunoterapia (RIT). Este estudo concentrou-se nas condições de conjugação do anticorpo monoclonal rituximab (MabThera&reg) com grupamentos quelantes bifuncionais DOTA e DTPA. Na marcação dos Acm conjugados com lutécio-177, foram estudadas as condições de pré-purificação do Acm, condições de conjugação, determinação de número de quelantes acoplados à molécula do anticorpo, purificação do anticorpo conjugado, radiomarcação do anticorpo conjugado, com lutécio-177, purificação do anticorpo marcado, a ligação específica in vitro dos compostos marcados às células Raji, e distribuição biológica em camundongos BALB/c sadios. As três metodologias empregadas na pré-purificação do anticorpo (diálise, cromatografia de exclusão molecular com coluna Sephadex G-50 e ultrafiltração) demonstram-se eficientes e proporcionaram recuperação da amostra superior a 90%. A metodologia de ultrafiltração foi considerada a mais simples e prática, podendo ser aplicada a procedimentos rotineiros de produção de radiofármacos. Além disso, proporcionou a recuperação final de amostra de 97% em microlitros. Nas conjugações do anticorpo com os quelantes DOTA e DTPA em razões molares diferentes do Acm:quelante, observou-se número de grupamentos quelantes acoplados à molécula do Acm proporcional à razão molar estudada. Quando foi avaliada a influência de condições diferentes de conjugação no número de quelantes acoplados à molécula do Acm, não foram observadas diferenças significativas, com resultados de pureza radioquímica (PR) inferior a 80% em todas as condições estudadas. Na comparação de métodos de purificação do Acm conjugado, a abordagem inédita apresentada neste estudo, na qual a cromatografia de exclusão molecular foi combinada com a ultrafiltração resultou em maior eficiência na purificação e preservação da estrutura do anticorpo. Nos estudos de radiomarcação do anticorpo conjugado com DOTA e DTPA, os imunoconjugados de DTPA apresentaram, de forma geral, maior eficiência de marcação com resultados reprodutíveis quando comparados com os imunoconjugados de DOTA, considerando-se as diferentes razões molares utilizadas. As metodologias cromatográficas empregadas no controle de pureza radioquímica do composto radiomarcado proporcionaram a discriminação das diferentes espécies radioquímicas no meio de marcação. A metodologia de purificação do composto conjugado e radiomarcado utilizada proporcionou a obtenção de compostos com alta pureza radioquímica, 97,4&plusmn;1,3% (DOTA 1:50) e 98,7&plusmn;0,2% (DTPA 1:50). Nos estudos de ligação específica às células tumorais Raji, o anticorpo conjugado com quelante DTPA nas razões molares de 1:50 e 1:20 apresentaram perfil semelhante de ligação, com aumento da porcentagem de ligação específica proporcional à concentração celular, enquanto que o imunoconjugado na razão molar de 1:10 apresentou alta porcentagem de ligação não específica. Os resultados obtidos nos estudos de biodistribuição in vivo do anticorpo conjugado e radiomarcado nem sempre se mostraram compatíveis com a biodistribuição de anticorpos radiomarcados íntegros. No caso do quelante DOTA, o imunoconjugado obtido a partir da razão molar 1:20, apresentou melhores características de biodistribuição. No caso do quelante DTPA, a razão molar utilizada pareceu refletir diretamente no clareamento sanguíneo do anticorpo e todas as razões molares utilizadas apresentaram instabilidade in vivo. / Lymphomas are malignancies or cancers that start from the malign transformation of a lymphocyte in the lymphatic system. Lymphomas are divided in two major categories: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Patient with NHL are generally treated with radiotherapy alone or combined with immunotherapy using monoclonal antibody rituximab (MabThera&reg). Currently, monoclonal antibodies (Mab) conjugated with bifunctional chelate agents and radiolabeled with metallic or lanthanides radionuclides are a treatment reality for patients with NHL by the principle of radioimmunotherapy (RIT). This study focused on the conditions of conjugation of Acm rituximab (MabThera&reg) with bifunctional chelating agents DOTA and DTPA and labeling with 177-luthetium. Various parameters were studied: method of Acm purification, conditions of Acm conjugation and the determination of the number of chelate coupled to the Acm, the purification of the conjugated Mab, labeling conditions with lutetium-177, purification of the radiolabeled immunoconjugate, radiochemical purity (RP), in vitro specific binding determination to Raji cells (Human Burkitt) and biological distribution performed in normal BALB/c mouse. The three methodologies employed in pre-purification of Acm (dialysis, size exclusion chromatograph and ultrafiltration) demonstrated to be efficient; they provided sample recovery exceeding 90%. However, the methodology of ultrafiltration resulted in greater sample recovery and in microliters. The number of chelate attached to the Mab molecule was proportional to the molar ratio studied. When the influence of different conditions of conjugation in the number of chelate bounded to the Mab was studied, no notable differences were observed. The RP < 80% was observed in all the methods applied. Purification of the conjugated antibody by different methods showed that the innovative combination of Sephadex and ultrafiltration methods resulted in higher efficiency of purification. The optimized conditions for purification of the conjugated antibody preserved the protein integrity. Radiolabelling studies of DOTA and DTPA immunoconjugated showed that DTPA derivatives presented, in general, radiochemical yield superior than DOTA conjugated Mab, considering the different molar ratios studied. The chromatographic methods employed in the RP determination were efficient to separate the different radiochemical species presented in the reaction medium. The methodology used in the purification of the labeled Mab resulted in labeled compounds with high radiochemical purity, 97.4&plusmn;1.3% (DOTA 1:50) and 98.7&plusmn;0.2% (DTPA 1:50). Considering specific cell binding assays (Raji cells), the Mab conjugated to DTPA at 1:50 and 1:20 molar ratios presented similar results, and the percent of cell binding were proportional to the cell concentration, whereas the cell binding for 1:10 molar ratio showed high percent of nonspecific cell binding. The results of in vivo biodistribution studies of labeled Mab not always were compatible with the biodistribution of intact radiolabelled antibody. The DOTA immunoconjugated produced at 1:20 molar ratio, showed better performance in biodistribution studies. In the case of DTPA immunoconjugated, the blood clearance seems to be influenced by the molar ratio applied and the immunoconjugated produced with DTPA chelate at different molar ratio resulted in high in vivo instability compounds.

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana. / Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis.

Esporotricose é uma micose subcutânea, causada principalmente pelo fungo Sporothrix schenckii e a nova espécie Sporothrix brasiliensis. Foi relatado na literatura que os camundongos infectados com a cepa de S. schenckii M-64 produziu uma resposta imune mista, em que o soro dos camundongos infectados reagiram apenas com uma glicoproteína de 70 kDa (gp70), identificada como importante fator de virulência. Assim o presente trabalho visa avaliar a importância à virulência e eficácia do anticorpo monoclonal P6E7 em outras cepas do complexo Sporothrix. Camundongos foram infectados com as cepas de S. schenckii (1099-18 e 15383) e S. brasiliensis (5110 e 17943). Cada 7 dias, o baço e fígado foram retirados para a análise do UFC e citocinas. Foi realizado western blot com o soro dos camundongos infectados e com anticorpo monoclonal P6E7 utilizando o exoantígenos das diferentes cepas. Foi realizado protocolo de tratamento com o anticorpo P6E7 nos camundongos infectados, para a avaliação da carga fúngica no baço e no fígado. Análise de citocinas mostrou que as cepas de S. schenckii induziram uma resposta mista Th1/Th2, entretanto nos camundongos infectados com as cepas de S. brasiliensis, não foi observada produção significativa de citocinas. No western blot realizado com exoantígenos das cepas de Sporothrix sp. foi observado diferentes componentes antigênicos, quando o soro de camundongos infectados foi utilizado, porém a reação com anticorpo monoclonal contra a gp70 demonstrou que a gp70 estava presente apenas no exoantígeno da cepa mais virulenta 5110. Infecção realizada com todas as cepas demonstrou cronicidade pela análise UFC. O tratamento com o AcMo P6E7 foi eficaz no tratamento da esporotricose experimental, sendo capaz de diminuir a carga fúngica, principalmente no baço dos camundongos infectados. / Sporotrichosis is a subcutaneous mycosis, caused mainly by fungi Sporothrix schenckii and the new specie Sporothrix brasiliensis. Was reported in previous studies that mice infected with low virulent S. schenckii M-64 strain, produced a mixed immune response, where the infected mice serum reacted only with a 70 kDa glycoprotein (gp70), identified as important virulence factor. Thus, the present study aims to evaluate the virulence importance and effectiveness of monoclonal antibody P6E7 in other Sporothrix complex strains. Mice were infected with Sporothrix yeast strain of S. schenckii (1099-18 and 15383) and S. brasiliensis (5110 and 17943). Each 7 days, the spleen and liver were taken for CFU and cytokines analysis. The western blot was performed with the serum obtained from infected mice and a monoclonal antibody P6E7 using exoantigens from different strains. Was performed treatment protocol with the monoclonal antibody P6E7 on infected mice, for spleen and liver fungal burden evaluation. Cytokine analysis showed that S. schenckii strains induce a mixed Th1/Th2 response, however in the S. brasiliensis strains wasn\'t observed significant cytokine production. In western blot accomplished with the strains exoantigens was observed different antigenic components when the infected mice serum was used, however with monoclonal antibody against gp70 demonstrated that gp70 was present only in most virulent strain 5110. Infection performed with all strains demonstrated chronicity for CFU analysis. Treatment with Mab P6E7 was effective in experimental sporotrichosis, with reducing capable of fungal load, mainly in the spleen of infected mice.

Evidências sugerem que o fator de crescimento de fibroblasto 2 (FGF2), produzido por melanomas, possui importante papel no crescimento tumoral, angiogênese e metástase. Assim, o uso de anticorpo monoclonal (mAb) que reconhece e bloqueia a atividade de FGF2 é uma abordagem a ser considerada em oncologia. O propósito desse estudo foi avaliar a aplicação diagnóstica e terapêutica de um novo anticorpo anti-FGF2, 3F12E7 IgG1, em melanoma experimental B16-F10. Para isso, camundongos C57Bl/6 foram implantados subcutaneamente (ou intravenosamente, para ensaios de metástase) com células de melanoma murino B16-F10 (5x105 células/animal). Quando tumores alcançaram 3-4 mm de diâmetro (ou 24 h pós-inóculo de células B16-F10, no caso de ensaios de metástase), camundongos foram tratados com anti-FGF2 3F12E7 IgG. Animais controle receberam igual volume do veículo ou quantidade de anticorpo controle de isotipo. Grupos: animais tratados com (1) anti-FGF2 3F12E7 IgG1; (2) ligante de CEA IgG1 (controle de isotipo); e (3) veículo. O tratamento dos camundongos portadores de tumor com anti-FGF2 IgG resultou, comparado com os controles salina e de isotipo, em uma redução no número de focos metastáticos nos pulmões (ANOVA, p < 0,05), em ensaios de metástase experimental, bem como em uma menor taxa de crescimento de tumores subcutâneos (n=7/grupo). Esse resultado é acompanhado por uma redução na densidade vascular do tumor, conforme determinado por imunomarcação para CD34 ou CD31. A captação tumoral de anti-FGF2 3F12E7 IgG foi avaliada por métodos de medicina nuclear, usando esse anticorpo radiomarcado com tecnécio-99m. Estudos SPECT/CT in vivo e de biodistribuição ex vivo revelaram que 99mTc-anti-FGF2 3F12E7 IgG pode atingir eficientemente tumores subcutâneos e metastáticos de B16-F10. Assim, esses dados sugerem que anti-FGF2 3F12E7 IgG pode ser uma estratégia antitumoral promissora para melanoma, bem como uma potencial ferramenta de imagem a ser explorada, atuando como um possível traçador para rastrear tumores FGF2-positivos e mapear esse estímulo angiogênico no microambiente tumoral. Aprovado pelo comitê de ética (CAPPesq): número 0942/09 / Compelling evidence suggests that fibroblast growth factor 2 (FGF2), produced by melanomas, plays important role in tumor growth, angiogenesis and metastasis. Therefore, the use of a monoclonal antibody (mAb) that recognizes and blocks FGF2 activity is seen as an approach to be considered in oncology. The purpose of this study was to evaluate the diagnostic and therapeutic application of a new anti-FGF2 antibody, 3F12E7 IgG1, in experimental melanoma B16-F10. For this, C57Bl/6 mice were subcutaneously (or intravenously, for experimental metastasis assay) implanted with murine melanoma B16-F10 cells (5x105 cells/animal). When tumors reached 3-4 mm in diameter (or 24 h after B16-F10 cells injection, in the case of metastasis assay), mice started receiving anti-FGF2 3F12E7 IgG. Control mice received equal volume of vehicle or isotype control IgG amount. Groups: (1) anti-FGF2 3F12E7 IgG1-treated, (2) CEA-binding IgG1-treated (isotype control) and (3) vehicle-treated mice. The treatment of tumor-bearing mice with anti-FGF2 IgG, compared with saline and isotype controls, led to a reduction in the number of metastatic foci in the lungs (ANOVA test, p < 0.05), in experimental metastasis assays, as well as to a lower subcutaneous tumor growth rate (n=7 per group). This result is accompanied by a reduction in the tumor vascular density, as determined by CD34 or CD31 staining. The anti-FGF2 3F12E7 IgG tumor uptake was evaluated by nuclear medicine approaches, using this antibody radiolabeled with technetium-99m. In vivo SPECT/CT and ex vivo biodistribution studies reveled that 99mTc-anti-FGF2 IgG could efficiently achieved B16-F10 subcutaneous and metastatic tumors. Thus, these data suggest that the anti-FGF2 3F12E7 IgG may be a promising antitumor strategy for melanoma, as well as a potential imaging tool to be explored, working as a possible tracer to identify FGF2-positive tumors and map this angiogenic stimulus in the tumor microenvironment. Ethics committee (CAPPesq) approval number 0942/09