Objectives: The aim of this thesis was to evaluate the clinical use of rituximab (RTX) in rheumatoid arthritis (RA) patients treated in routine clinical practice in the UK, taking account of the previous therapies (anti-tumour necrosis factor (anti-TNF) therapies).Methods: The analysis involved RA patients registered with the British Society for Rheumatology Biologics Register. Kaplan-Meier survival analysis was used to study the persistence with anti-TNF therapies. Drug persistence was compared across the anti-TNF therapies and according to the most common concomitant non-biological disease modifying anti-rheumatic drugs (nbDMARDs) for up to five years. Adjusted multivariate Cox proportional hazard models were used to compare drug persistence across the subgroups. Change in Disease Activity Score (DAS28) and European League Against Rheumatism (EULAR) response were used to assess the clinical effectiveness of RTX while change in Health Assessment Questionnaire (HAQ) score was used to assess the physical function of the patients six months after starting RTX. Logistic regression was used to compare EULAR response and achieving a minimal clinically important difference (MCID) in HAQ (at least 0.22) between patients who started RTX and those who switched to a second alternative anti-TNF after failing a first anti-TNF therapy. Multivariate regression analyses were used to identify factors associated with the clinical effectiveness and the improvements in physical function six months after starting RTX. The models included baseline demographics, disease characteristics, baseline quality of life and previous drug history. Results: Among 10,396 RA patients receiving their first anti-TNF therapy, five-year drug persistence (95% CI) was 42% (41%: 43%). Infliximab was the most likely discontinued anti-TNF therapy. Compared to methotrexate (MTX), patients receiving no nbDMARD, leflunomide or sulfasalazine were more likely to discontinue their first anti-TNF therapy while patients receiving MTX in combination with other nbDMARDs showed superior persistence with their anti-TNF therapy. After failing the first anti-TNF therapy, patients who switched to RTX were significantly more likely to achieve EULAR response and MCID in HAQ; odds ratio (95% CI) 1.31 (1.02: 1.69) and 1.49 (1.07: 2.08) respectively compared to those who switched to an alternative anti-TNF therapy. In a cohort of 646 RA patients who started RTX, the mean DAS28 scores significantly improved six months after starting RTX with mean (95% CI) change of -1.42 (-1.53: -1.30). 17% of the patients achieved a good EULAR response and 43% achieved a moderate response. Higher baseline DAS28 score and positive rheumatoid factor (RF) status were significantly associated with a decrease in disease activity, while females and patients with higher baseline HAQ score were less likely to respond to RTX. In a cohort of 484 patients receiving RTX, the mean HAQ scores significantly improved by -0.12 (-0.16: -0.09) units. High baseline HAQ score was significantly associated with six months improvement in HAQ. Older patients, females, current smokers and patients receiving concurrent steroids were less likely to show an improvement in their HAQ scores. Conclusions: Compared to MTX, concomitant use of two or three nbDMARDs combinations including MTX with anti-TNF therapies resulted in better persistence with the anti-TNF therapies. After failing the first anti-TNF therapy, starting RTX may be of more benefit than switching to an alternative anti-TNF therapy. RTX has proven to be effective in improving both the clinical and patients' reported outcomes in routine clinical practice in the UK. Response to RTX was influenced by baseline DAS28, RF status, baseline HAQ, age, gender, smoking, and concurrent use of steroids.

Introduktion Granulomatös polyangit (Wegener) (GPA (WG)) är en allvarlig sjukdom som orsakar inflammation på insidan av kroppens små blodkärl. Behandling utgörs av höga doser kortison samt cyklofosfamid. När sjukdomen är under kontroll ersätts cyklofosfamid av andra, mildare läkemedel. Benägenhet för återfall och med det ökad risk för allvarliga biverkningar av cyklofosfamid gör att behovet av säkrare behandlingsalternativ är stort. Syfte Syftet med denna studie var att utvärdera det biologiska läkemedlet rituximab med avseende på indikation för behandling, effekt och säkerhet vid underhållsbehandling av patienter med återkommande GPA (WG).   Material och metoder Retrospektiv insamling av journaldata. Patienter diagnosticerade med GPA (WG) enligt vedertagna kriterier, vårdade på reumatologiska kliniken vid Akademiska sjukhuset, som fått minst en infusion rituximab inkluderades och följdes till och med 2011-11-30. Resultat Elva patienter inkluderades varav sex var kvinnor. För tio av patienterna var behandlingsindikationen terapivikt med tidigare behandling. Medianåldern vid diagnos var 46 år och uppföljningstiden i genomsnitt 28 månader. Patienterna hade haft i genomsnitt tre återfall innan rituximab och antal olika läkemedel i genomsnitt drygt fem. Nio patienter har fått minst tre kurer rituximab och alla har svarat på behandlingen varav sju inte har några symptom från sin GPA (WG). Ur säkerhetssynpunkt var rituximab vältolererat med drygt fyra oönskade händelser per patient och år. Konklusion Resultaten av denna studie indikerar att rituximab kan fungera som underhållsbehandling vid GPA (WG) med benägenhet för återfall. Resultaten begränsas av att rituximabs effekt i förhållande till konventionell underhållsbehandling inte har studerats. Inte heller har potentiella risker vid långtidsexponering kunnat utvärderas.

Background: Burkitt lymphoma (BL) is a rare, highly aggressive B-cell malignancy treated most successfully with brief-duration, high-intensity chemotherapeutic regimens. The benefit of the addition of rituximab to these regimens remains uncertain. We sought to examine the effectiveness of chemotherapy with and without rituximab in patients with BL. Methods: This study is a retrospective cohort study of all adult patients with BL diagnosed and treated with modern, dose-intense chemotherapeutic regimens from 1998–2008 at two tertiary care institutions. All cases were confirmed by application of WHO 2008 criteria by hematopathologists. Medical records were reviewed for patient-, disease-, and treatment- related factors as well as treatment response and survival. Factors associated with survival were analyzed using Cox proportional hazards modeling. Results: A total of 35 patients were analyzed: 18 patients received rituximab with chemotherapy (R-chemo) and 17 received chemotherapy (chemo) alone. The median age was 42 (range 20–74 years); 57% were male; 71% had Ann Arbor Stage IV disease; 33% had central nervous system involvement; 78% had an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1. R-chemo was associated with significantly longer overall survival (OS) than chemo alone (5-year OS 70% and 29%, respectively, p = 0.040). On multivariate regression analysis, poor performance status and central nervous system involvement were associated with poorer survival. The addition of rituximab to chemotherapy was associated with improved OS in patients with Burkitt lymphoma. Poor performance status and central nervous system involvement were prognostically significant on multivariate analysis.

Die zentrale Rolle der B-Zellen in der Pathogenese von Autoimmunerkrankungen hat in den letzten Jahren zu unterschiedlichen therapeutischen Ansätzen geführt, B-Zellen direkt oder indirekt zu targetieren. Ein Beispiel hierfür stellt der monoklonale anti-CD20 Antikörper Rituximab dar. Derzeit ist wenig über das Regenerationsverhalten von B-Zellen nach Therapie mit Rituximab bekannt. Daher untersuchten wir die frühe Regnerationsphase und die Veränderungen des B-Zellrepertoirs. Am Beispiel der VH4 Familie der Immunglobulin schweren Ketten analysierten wir die Modulation des Immunglobulinrezeptor Repertoires durch die passagere B-Zelldepletion. Insgesamt wurden bei 5 Patienten 3 Zeitpunkte analysiert: vor Therapie, in der frühen Regenerationsphase (ERP- early regeneration period, mit einem B-Zellanteil > 1% im peripheren Blut) und in der späten Regenerationsphase (LRP- late regeneration period, 2-3 Monate nach der frühen Regenerationsphase). Bei 3 Patienten (A-C) wurden die Ig-VH4 Gene aus genomischer DNA amplifiziert und zu o.g. Zeitpunkten analysiert. Bei weiteren 2 Patienten (D und E) erfolgte die Analyse der Ig Gene in einzelnen B-Zellen mittels Einzelzellsortierung und Einzelzell RT-PCR. Die B-Zellregeneration nach Therapie mit Rituximab zeigte ein charakteristisches Regenerationsmuster mit einer Dominanz von unreifen CD10+ B-Zellen und CD38hi Plasmazellen während der frühen Phase der B-Zellrekonstitution. Im weiteren Verlauf kam es zu einer Abnahme dieser Zellen und einem Anstieg von naiven B-Zellen. Auf der molekularen Ebene zeigte sich vor und nach B-Zelldepletion eine unterschiedliche Nutzung der Ig-VH4 Gene. Mini Gene wie VH4-34 und VH4-39, die in Verbindung mit Autoimmunität stehen, waren vor Einleitung der Therapie überexprimiert. Durch die Behandlung mit Rituximab kam es zu einer Veränderung des Repertoires der regenerierenden B-Zellen mit einer reduzierten Benutzung der VH4-39 Gene im B-Zellpool. Tief greifende Veränderungen fanden sich im regenerierenden Repertoire, mit einem relativen Anstieg von stark mutierten (>=9 Mutationen / Ig Sequenz) B-Zellen.. Die Immunphänotypisierung zeigte, dass diese hochmutierten B-Zellen den Ig-klassengeswitchten Gedächtnis B-Zellkompartiment, insbesondere den Plasmazellen zughörig sind. Um diese Hypothese zu untermauern, erfolgte bei 2 Patienten eine Einzelzellsortierung dieser Plasmazellen während der frühen Regenerationsphase, welche einen vergleichbaren Mutationsstatus zeigte. Da Plasmazellen kein CD20 Molekül exprimieren, werden sie durch eine Therapie mit Rituximab nicht direkt eliminiert. Allerdings zirkulieren sie nicht im peripheren Blut während der Phase der B-Zelldepletion. Während der frühen Regenerationsphase (ERP) lassen sie sich in der Peripherie erneut nachweisen. Es wurde deshalb untersucht ob auch Plasmazellen durch die Therapie moduliert werden, obwohl sie nicht direkt durch Rituximab targetiert werden. In diesem Zusammenhang erfolgte eine detaillierte Analyse des Mutationsmusters der Plasmazellen vor Therapie und während der frühen Regenerationsphase. Die Analyse der Mutationshäufigkeit in RGYW/WRCY Hotspot Motive (R=purine, Y=pyrimidine, W=A/T) erlaubt Abschätzung in wieweit die somatische Hypermutation der B-Zellen durch T-Zell abhängige Differenzierung erfolgte. Die Plasmazellen vor Therapie zeigten einem verminderten Targeting der RGYW/WRCY Motive. Im Gegensatz hierzu zeigte sich in den rezirkulierenden Plasmazellen während der frühen Regenerationsphase ein zunehmendes Targeting der RGYW/WRCY Motive. Dies spricht für einen Repertoire Shift zu mehr T-Zellabhängigen B-Zell Mutation. Ein Zusatand, wie er bei Gesunden beobachtet wird. Um die Hypothese der Rituximab-induzierten Plasmazell Modulation zu stützen wurde die R/S- Ratio (replacement to silent mutations ratio) der hypervariablen Regionen (CDRs) der Plasmazell Ig Sequenzen bestimmt. In unserer Studie war die mittlere R/S Ratio der CDRs der Plasmazellen vor Therapie entsprechend relativ niedrig (1.87). Interessanterweise kam es in der frühen und späten Regenerationsphase zu einer signifikant erhöhten R/S Ration in den rezirkulierenden Plasmazellen mit Werten von 2.67 bzw. 3.60. Die verminderte R/S Ratio in den CDRs der Plasmazellen kann als Entwicklung des Ig-Repertoires durch positive Antigenselektion interpretiert werden und weist damit eine Therapie induzierte Veränderung auf, die dem entspricht wie man sie bei Gesunden findet. Zusammenfassend zeigt unsere Studie, dass die passagere B-Zelldepletion mit Rituximab zu einer Modulation des Plasmalzellkompartimentes führt, welches nicht direkt durch die Therapie targetiert wird. Die Modulation der Plasmazellen bei der RA kann eventuell auch als möglicher Biomarker entwickelt werden, um ein Ansprechen auf die Therapie vorherzusagen. Dies muss im Weiteren untersucht werden, um tiefer greifende Einblicke in Prozesse zu erlangen, die durch zukünftige Therapien beeinflussbar werden. / B cells play diverse roles in the immunopathogensis of autoimmune diseases several approaches targeting B cell directly or indirectly are in clinical practice in the treatment of autoimmunity. In this regard, temporal B cell depletion by rituximab (anti CD20 antibody) is being appreciated and gaining more importance in recent years. To date, little is known about the regeneration profile of B cells following B cell depletion. We wanted to investigate the early replenishing B cells and examine the dynamic changes in the repertoire. we studied the immunoglobulin receptor (IgR) modulation of Ig-VH4 genes as representative of the heavy chain family. Five patients were included in the study and therapy induced alterations were assessed. Three time points namely before therapy, early regeneration phase (ERP- the early time point during regeneration where just above 1% B cells were found in the peripheral lymphocyte pool) and later regeneration phase (LRP- which commenced 2-3 months following ERP) were chosen. In three patients (A-C), Ig-VH4 genes were amplified from total genomic DNA during the above-mentioned all time points and in another two patients (D and E), Ig genes during ERP were studied by single cell amplification technique. Firstly, B cell regeneration followed the characteristic regeneration pattern as reported by several groups, with a predominant circulation of CD38hi expressing plasma cells and immature B cells in the ERP. During LRP, the proportion of these cells reduced relatively and the levels of naïve B cells rose gradually. On a molecular level, Ig-VH4 variable gene usage prior and post B cell depletion was determined and it was noticed that a diverse set of Ig-VH4 genes were employed in the repertoire before and after therapy. Mini gene segments such as VH4-34 and VH-4-39, which were reported to be connected with autoimmunity, were over expressed in the B cell repertoire before therapy. Profound changes were noticed in the early reemerging repertoire with a relatively increased population of intensely mutated B cells. These B cells acquired >=9 mutations in the Ig genes. Immunophenotyping with specific surface markers revealed that these highly mutated B cells evolve from the isotype-switched memory compartment especially the plasma cells. To support the hypothesis that the highly mutated B cells observed during ERP were plasma cells we carried out single cell amplification of individual plasma cells in another two patients during ERP and compared the mutational load, which remained similar. Actually plasma cells do not express CD20 on their surface and are not eliminated by rituximab therapy. However they were not observed in the peripheral blood following B cell depletion. The earliest time point when plasma cells are found again in peripheral circulation is the early recovery period (ERP). Therefore, it was intriguing to ascertain if the plasma cells were also modulated by rituximab therapy although they were not directly targeted by the therapy. We investigated if there is a therapy mediated mutational modulation of the plasma cells though these are not directly targeted by the therapy. We examined the confinement of mutations to the pre-defined RGYW/WRCY hotspot motifs (R=purine, Y=pyrimidine, W=A/T) in the plasma cells, which provides information on the involvement of T cells in B cell somatic hypermutation (SHM). Plasma cells before rituximab manifested the characteristics of active disease, which was revealed by restricted mutational targeting to the RGYW/WRCY motifs. The reemerging plasma cells during ERP had an increased targeting of the RGYW/WRCY motifs which indicated for a more pronounced T cell mediated B cell mutations which is the scenario observed in the healthy subjects. To further support the hypothesis of rituximab-mediated plasma cell modulation, we delineated the replacement to silent mutations ratio (R/S) in the hypervariable regions (CDRs) of the plasma cell Ig sequences. Within our study, the mean R/S ratio in the plasma cell CDRs of the patient group was relatively low (1.87) before rituximab treatment and interestingly this ratio increased significantly in the recirculating plasma cells to values of 2.67 and 3.60 in ERP and LRP status respectively. The increase in R/S ratios in reemerging plasma cells can be interpreted as a shaping of the Ig-repertoire by positive antigen selection as seen in healthy individuals. To conclude, our study demonstrates temporal B cell depletion by rituximab therapy seems to modulate also the plasma cell compartment, which is not directly targeted by the therapy. Modulation of plasma cells in RA could be also used as a potential biomarker in studying the effective response in RA treatment. This needs to be further explored to gain deeper insights into the underlying processes, which may be influenced by future therapies.

L'evolució clínica de la leucèmia limfocitària crònica (LLC) és amb freqüència indolent, però és una malaltia que roman incurable. Els pacients simptomàtics tenen una supervivència entre 1-7 anys, fins i tot utilitzant les millors opcions terapèutiques. Encara que han estat avaluades tot una sèrie de combinacions terapèutiques en pacients amb recaiguda o refractaris als tractaments, cap alternativa ha demostrat ser superior. Per tant, és evident la necessitat de trobar alternatives terapèutiques per aquests pacients. Diferents estudis en fase II han mostrat que l'associació del rituximab a la quimioteràpia ha millorat el percentatge de resposta i el període lliure de malaltia. No obstant, tots els pacients a la llarga experimenten una progressió de la malaltia. Un dels principals mecanismes d'acció del rituximab és la citotoxicitat cel·lular depenent d'anticossos. Les principals cèl·lules efectores implicades en aquest mecanisme d'acció són les cèl·lules portadores de receptors Fcγ, i dintre d'aquest grup les que juguen un paper primordial són les cèl·lules NK. La hipòtesi general va ser l'anàlisi de molècules potenciadores de la capacitat citotòxica de les cèl·lules NK i de l'ADCC, per poder ser utilitzades associades al rituximab com a adjuvants en el tractament de les leucèmies limfàtiques cròniques. Entre aquestes molècules es va estudiar el CpG ODN A per mimetitzar l'activitat estimuladora del DNA bacterià, a través del TLR9 i provocar una forta activació i síntesi d'IFNγ per cèl·lules NK. L'altre molècula va ser l'IL-15 per estar estretament relacionada en la supervivència, proliferació i activació de les cèl·lules NK. Es va observar que la IL-15 i el CpG ODN A eren 2 molècules estimuladores que potenciaven significativament la capacitat citotòxica de PBMCs, en presència o absència de rituximab, quan s'enfrontaven tant a una línia cel·lular de limfoma B humà com a cèl·lules leucèmiques de LLC-B. La principal població efectora responsable d'aquesta potenciació citotòxica van ser les cèl·lules NK. No obstant el mecanisme d'acció responsable va ser diferent. Mentre que la IL-15 incrementava la capacitat citotòxica de la cèl·lula NK tant directament com indirectament, el CpG ODN A ho feia només indirectament requerint de senyals addicionals presents a les PBMCs. Quan les PBMCs es van enfrontar a cèl·lules de limfoma B de la línia cel·lular Raji va resultar que els dos estímuls es comportaven igual. Però quan van ser cèl·lules leucèmiques de LLC-B, la IL-15 es va comportar com un estímul significativament més potent que el CpG ODN A incrementant la citotoxicitat natural i l'ADCC. La IL-15 en aquest sentit va actuar incrementant l'expressió de receptors relacionats amb cèl·lules NK en desgranulació (CD16, CD69 i NKG2D) i del receptor LFA-1 implicat en la senyalització de la citotoxicitat (adhesió cel·lular). Així com produint un augment significatiu d'IFN-γ. En presència de TGF-β, citocina immunosupressora que disminueix de manera significativa la citotoxicitat natural i l'ADCC de PBMCs enfrontades a cèl·lules leucèmiques de LLC, la IL-15 va poder contrarestar aquest efecte supressor. De la mateixa manera en presència d'IL-15, la disminució que provoca el TGF-β en la producció d'IFN-γ, va ser menor i més variable no sent significativa. / The clinical course of chronic lymphocytic leukemia is often insidious, but it is a disease that remains not treatable. Even using the best therapeutic options, symptomatic patients have a survival between 1-7 years. Several series of therapeutic combinations have been evaluated for patients with relapses and refractory to treatment, but none has been demonstrated to be superior. Therefore, it is notable the need to find therapeutic alternatives for these patients. Different studies in phase II have shown that the addition of rituximab to chemotherapy has improved the percentage of response and the disease free period. However, all patients suffer progression of the disease at long term. One of the main mechanisms of action of rituximab is the antibody mediated cytotoxicity. The most important effector cells involved in this mechanism of action are Fcγ receptors cells, specifically NK cells. The general hypothesis is the analysis of molecules enhancing the cytotoxic capacity of NK cells and ADCC, for them to be used in addition to rituximab as adjuvant in the treatment of chronic lymphocytic leukemia. Amongst these molecules, CpG ODN A was studied because its capacity to mimetise the stimulating activity of bacterial DNA through TLR9 and to induce a strong activation and synthesis of IFNγ by NK cells. The other molecule was IL-15, due to its involvement in survival, proliferation and activation of NK cells. It was observed that IL-15 and CpG ODN A were two stimulating molecules than significantly enhanced the cytotoxic capacity of PBMCs, in the presence or absence of rituximab, when they were confronted to cells from B lymphoma Raji line and to leukemic cells from CLL-B. The major effector population responsible for this cytotoxic enhancement are the NK cells. However, the mechanism of action is different. Whereas IL-15 increased the cytotoxic capacity of the NK cell directly as much as indirectly, CpG ODN A did it only indirectly, requiring of additional signals present in PBMCs. When PBMCs were confronted to Raji cells from B cell lymphoma, the two stimuli had a similar behavior. However, when PBMCs were confronted to leukemic cells from CLL-B, Il-15 was a stronger stimulus than CpG ODN A, enhancing the natural cytotoxicity and ADCC. In this way, IL-15 worked increasing the expression of receptors related to degranulating NK cells (CD16, CD69 and NKG2D) and of LFA-1 receptor, implied in the cytotoxicity signaling (cellular adhesion), and increasing significantly IFNγ production. In the presence of TGF-β, immunosupressor cytokine that decrease significantly the natural cytotoxicity and ADCC of PBMCs confronted to leukemic cells from CLL, IL-15 is able to counteract this suppressive effect. In the same way, the reduction caused in IFN-γ production is going to be smaller and more variable in the presence of IL-15, although not significantly.

CD20 is a 35kDa surface antigen expressed on B cells from the early pre-B stage through the mature B stage. Moreover, the CD20 antigen is found on a majority of B cell malignancies. Rituximab is a chimeric anti-CD20 monoclonal antibody which has been extensively used alone or in combination in the treatment of CD20+ B cell malignancies including acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL), as well as in the treatment of numerous autoimmune disorders. Despite its emerging use in the clinic, 30% to 50% of patients with low-grade NHL exhibit no clinical response to Rituximab. Previous work to elucidate the mechanisms of Rituximab resistance has established that antibody dependent cellular cytotoxicity (ADCC) is important as a predominant mechanism of lymphoma cell clearance and that Fcγ receptors (FcγRs) are critical for the in vivo actions of Rituximab in NHL. Natural killer group 2D, NKG2D is a major activating receptor on T lymphocytes and natural killer cells. The NKG2D–ligand (NKG2D-L) interaction triggers an activating signal which results in cytotoxic lysis of the cell expressing the ligand. One potential ligand for murine NKG2D is the retinoic acid early 1β (Rae-1β) protein which is expressed during cellular stress and has a high affinity for the NKG2D receptor in mice. We have recently shown that an anti-HER2-IgG3 fused to murine NKG2D Ligand, Rae1β inhibited HER2+ tumor growth significantly more than Herceptin alone. Similarly, our objective is to enhance the performance of anti-CD20 directed therapy through activation of NK cells by an anti-CD20 antibody encoding the same NK activation ligand. Previous results with anti-HER2-IgG3-Raelβ led us to hypothesize that a CD20 specific fusion protein will bind to CD20 expressing tumor cells and deliver an activation signal to local NKG2D receptors on effector cells triggering a non-FcγR dependent anti-tumor response. Here we show that anti-CD20-NKG2D-L can be synthesized and tested for its ability to bind human CD20 and activate NK cells through the NKG2D receptor in vitro.

Linfomas são cânceres provenientes da transformação de um linfócito no sistema linfático, sendo que, o mais comum é o Linfoma Não-Hodgkin (LNH). Avanços na imunologia e na biologia molecular têm auxiliado na detecção desses tumores e aberto caminhos para novas estratégias de tratamento, como a Radioimunoterapia. O rituximab é um anticorpo monoclonal quimérico anti-CD20 já utilizado como imunoterápico no tratamento de LNH refratários ou recidivos. O objetivo deste trabalho foi estudar a conjugação deste anticorpo ao quelante bifuncional DOTA-NHS-éster e radiomarcar este imunoconjugado com o radioisótopo 177Lu, com o objetivo de desenvolver um radioimunoterápico para tratamento de LNH. Estudos de imunoconjugação com diferentes razões molares rituximab:DOTA foram estudadas (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 e 1:1000) afim de avaliar qual condição conferia maior pureza radioquímica. A estabilidade dos imuconjugados foi analisada por cromatografia de alta eficiência por até 240 dias em diferentes condições de armazenamento. A estabilidade do imuconjugado radiomarcado foi avaliada após incubação a 2-8 °C e em soro humano a 37 °C e a ligação às proteínas séricas foi determinada. Estudos de biodistribuição foram realizados em camundongos Swiss sadios a fim de caracterizar biologicamente o imunoconjugado radiomarcado. Com o objetivo de analisar se os processos de conjugação e de radiomarcação não danificaram a capacidade de reconhecimento do antígeno (imunorreatividade) deste anticorpo, realizou-se estudos preliminares de ligação às células de LNH (Raji). Os imuconjugados de razão molar baixa (1:5, 1:10 e 1:20) mostraram-se estáveis quando armazenados por até 240 dias em diferentes condições. A análise em cromatografia em camada delgada e CLAE, revelou que o Acm conjugado na razão molar 1:50 foi radiomarcado com alta pureza radioquímica (superior a 95%) quando purificado em coluna PD-10. Este mesmo radioimunoconjugado apresentou razoável estabilidade a 2-8° C. A análise da estabilidade em soro humano não indicou grande metabolismo pelas enzimas do soro. O radioimuconjugado apresentou alta ligação às proteínas séricas indicando clareamento sanguíneo lento, o qual foi confirmado pelos estudos in vivo. O radioimunoconjugado apresentou alta captação no fígado o que é característico de anticorpos monoclonais. Os estudos preliminares de competição indicaram que o processo de obtenção do radioimunoconjugado não prejudicou sua ligação às células Raji sendo esta ligação específica. / Lymphomas are tumors origened from the transformation of a lymphocyte in the lymphatic system. The most common lymphoma is the Non-Hodgkin Lymphoma (NHL). Advances in immunology and molecular biology have been improving NHLs detection and treatment strategies development, such as Radioimmunotherapy (RIT). Rituximab is an anti-CD20 monoclonal antibody used as immunotherapeutic to treat refractory or relapsed NHL. The goal of the present work was to conjugate this antibody to DOTA-NHS-ester bifunctional chelator and to radiolabel it with 177Lu radioisotope in order to develop a radioimmunotherapeutic agent for NHLs treatment. Different rituximab to DOTA molar ratios (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 and 1:1000) were evaluated in order to determine the best condition for obtaining the highest radiochemical purity of radioimmunotherapeutic. The stability of the unlabeled immunoconjugated was evaluated by high performance liquid chromatography (HPLC) for up to 240 days in different storage conditions. The stability of the labeled preparations was evaluated either after storing at 2-8 °C or incubation in human serum at 37 °C. The binding to serum proteins was also determined. In vivo studies were performed in healthy Swiss mice, in order to characterize the biological properties of labeled conjugate. Finally, preliminary studies of radioimmunoconjugated competitive binding to CD20 positive Raji cells were carried out in order to analyze if the process of conjugation and radiolabeling compromises the immunoreactivity of the antibody. The conjugation applying lower antibody to chelator molar ratios (1:5, 1:10 and 1:20) showed high stability when stored for up to 240 days in different conditions. The HPLC analysis showed that the monoclonal antibody conjugated in molar ratio 1:50 was labeled with higher radiochemical purity (> 95%) when purified in PD-10 column. This conjugate showed reasonable stability at 2-8 ° C. The analysis of the stability in human serum did not suggest high metabolic degradation by serum enzymes. The labeled conjugate showed high serum protein binding, suggesting slow blood clearance, which was confirmed by in vivo studies. The labeled conjugate presented high uptake in the liver, in accordance to biodistribution pattern of monoclonal antibodies. The preliminary competitive binding studies indicated a specific binding and suggest that the synthesis of 177Lu-DOTA-rituximab did not compromise its binding to CD20 positive tumor cells.

Linfomas são cânceres provenientes da transformação de um linfócito no sistema linfático, sendo que, o mais comum é o Linfoma Não-Hodgkin (LNH). Avanços na imunologia e na biologia molecular têm auxiliado na detecção desses tumores e aberto caminhos para novas estratégias de tratamento, como a Radioimunoterapia. O rituximab é um anticorpo monoclonal quimérico anti-CD20 já utilizado como imunoterápico no tratamento de LNH refratários ou recidivos. O objetivo deste trabalho foi estudar a conjugação deste anticorpo ao quelante bifuncional DOTA-NHS-éster e radiomarcar este imunoconjugado com o radioisótopo 177Lu, com o objetivo de desenvolver um radioimunoterápico para tratamento de LNH. Estudos de imunoconjugação com diferentes razões molares rituximab:DOTA foram estudadas (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 e 1:1000) afim de avaliar qual condição conferia maior pureza radioquímica. A estabilidade dos imuconjugados foi analisada por cromatografia de alta eficiência por até 240 dias em diferentes condições de armazenamento. A estabilidade do imuconjugado radiomarcado foi avaliada após incubação a 2-8 °C e em soro humano a 37 °C e a ligação às proteínas séricas foi determinada. Estudos de biodistribuição foram realizados em camundongos Swiss sadios a fim de caracterizar biologicamente o imunoconjugado radiomarcado. Com o objetivo de analisar se os processos de conjugação e de radiomarcação não danificaram a capacidade de reconhecimento do antígeno (imunorreatividade) deste anticorpo, realizou-se estudos preliminares de ligação às células de LNH (Raji). Os imuconjugados de razão molar baixa (1:5, 1:10 e 1:20) mostraram-se estáveis quando armazenados por até 240 dias em diferentes condições. A análise em cromatografia em camada delgada e CLAE, revelou que o Acm conjugado na razão molar 1:50 foi radiomarcado com alta pureza radioquímica (superior a 95%) quando purificado em coluna PD-10. Este mesmo radioimunoconjugado apresentou razoável estabilidade a 2-8° C. A análise da estabilidade em soro humano não indicou grande metabolismo pelas enzimas do soro. O radioimuconjugado apresentou alta ligação às proteínas séricas indicando clareamento sanguíneo lento, o qual foi confirmado pelos estudos in vivo. O radioimunoconjugado apresentou alta captação no fígado o que é característico de anticorpos monoclonais. Os estudos preliminares de competição indicaram que o processo de obtenção do radioimunoconjugado não prejudicou sua ligação às células Raji sendo esta ligação específica. / Lymphomas are tumors origened from the transformation of a lymphocyte in the lymphatic system. The most common lymphoma is the Non-Hodgkin Lymphoma (NHL). Advances in immunology and molecular biology have been improving NHLs detection and treatment strategies development, such as Radioimmunotherapy (RIT). Rituximab is an anti-CD20 monoclonal antibody used as immunotherapeutic to treat refractory or relapsed NHL. The goal of the present work was to conjugate this antibody to DOTA-NHS-ester bifunctional chelator and to radiolabel it with 177Lu radioisotope in order to develop a radioimmunotherapeutic agent for NHLs treatment. Different rituximab to DOTA molar ratios (1:5, 1:10, 1:20, 1:50, 1:250, 1:500 and 1:1000) were evaluated in order to determine the best condition for obtaining the highest radiochemical purity of radioimmunotherapeutic. The stability of the unlabeled immunoconjugated was evaluated by high performance liquid chromatography (HPLC) for up to 240 days in different storage conditions. The stability of the labeled preparations was evaluated either after storing at 2-8 °C or incubation in human serum at 37 °C. The binding to serum proteins was also determined. In vivo studies were performed in healthy Swiss mice, in order to characterize the biological properties of labeled conjugate. Finally, preliminary studies of radioimmunoconjugated competitive binding to CD20 positive Raji cells were carried out in order to analyze if the process of conjugation and radiolabeling compromises the immunoreactivity of the antibody. The conjugation applying lower antibody to chelator molar ratios (1:5, 1:10 and 1:20) showed high stability when stored for up to 240 days in different conditions. The HPLC analysis showed that the monoclonal antibody conjugated in molar ratio 1:50 was labeled with higher radiochemical purity (> 95%) when purified in PD-10 column. This conjugate showed reasonable stability at 2-8 ° C. The analysis of the stability in human serum did not suggest high metabolic degradation by serum enzymes. The labeled conjugate showed high serum protein binding, suggesting slow blood clearance, which was confirmed by in vivo studies. The labeled conjugate presented high uptake in the liver, in accordance to biodistribution pattern of monoclonal antibodies. The preliminary competitive binding studies indicated a specific binding and suggest that the synthesis of 177Lu-DOTA-rituximab did not compromise its binding to CD20 positive tumor cells.

CD20 est une cible thérapeutique validée pour l’immunothérapie des néoplasmes lymphoïdesdes cellules B, incluant la Leucémie Lymphoïde Chronique (LLC). Nous avons comparé les effets de rituximab et de GA101 (nouvel anticorps anti-CD20) contre les cellules LLC fraiches in vitro. Le marquage avec Annexine V a démontré une induction de l’apoptose après l’exposition au rituximab et GA101.Contrairement au rituximab, GA101 induisait une réduction du potentiel transmembranaire mitochondrial, uneffet qui peut être partiellement inhibé par la cyclosporine A et qui est partiellement caspase-dépendant. GA101induisait aussi la production des espèces d’oxygènes réactives. L’analyse du niveau d’expression des protéinespro- et anti-apoptotiques après exposition aux anticorps a démontré une forte hétérogénéité entre les échantillons.Bax subissait une activation de conformation et une translocation mitochondriale suite à l’exposition aux anticorps d’une manière caspase-indépendante. GA101, mais pas rituximab, induisait le clivage des caspase-8, -9et -3. En transfectant les cellules LLC avec un siRNA ciblant Bcl-xL utilisant la sonoporation, nous avons trouvéque la réduction du niveau d’expression de Bcl-xL est associée à une augmentation de la sensibilité aux anticorps. Nos résultats suggèrent que les voies de signalisation apoptotiques diffèrent entre rituximab et GA101avec une implication de la voie mitochondriale avec le GA101. L’inhibition de Bcl-xL peut constituer une façon pour sensibiliser les cellules LLC aux effets apoptotiques des anticorps anti-CD20. / CD20 is a validated target for the immunotherapy of B lymphoid neoplasms, including ChronicLymphocytic Leukemia (CLL). We compared the activities of rituximab and GA101 (novel anti-CD20 antibody)on fresh human CLL cells in vitro. AnnexinV staining demonstrated induction of apoptosis after exposure torituximab or GA101. Unlike rituximab, GA101 induced a reduction of the mitochondrial transmembranepotential, an effect which could be partially inhibited by cyclosporin A and which was partially caspasedependent.GA101 was also found to induce the production of Reactive Oxygen Species. Analysis of pro- andanti-apoptotic protein content after exposure to antibodies demonstrated a strong degree of heterogeneity between samples. Bax underwent conformational activation and mitochondrial translocation upon exposure toantibodies in a caspase-independent manner. GA101 but not rituximab induced cleavage of caspase-8, -9 and -3.By transfecting CLL cells with anti-Bcl-xL siRNA using a sonoporation method, we found that reduction of BclxLcontent was associated with increased sensitivity to these antibodies. Our results suggest that apoptoticsignalization pathways differ between rituximab and GA101 with a greater involvement of the mitochondrialpathway for GA101. Inhibition of Bcl-xL could constitute an approach to sensitize CLL cells to the apoptoticeffects of anti-CD20 antibodies.

Background: Epstein-Barr virus(EBV)-associated posttransplant lymphoproliferative disease (PTLD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). Especially in cases with involvement of the central nervous system (CNS) treatment is difficult because the efficacy of most chemotherapeutic agents as well as EBV-specific cytotoxic donor T cells in liquor is uncertain. In the last years the anti-CD20 monoclonal antibody Rituximab was intensively investigated in the treatment of EBV-PTLD. However, only 8 patients with B-cell lymphoma and CNS involvement treated with Rituximab were reported. Case Report: A 24-year-old female patient with acute T-lymphoblastic leukemia in second complete remission had received allogeneic, unrelated, T-cell depleted HSCT. 10 months later an EBV-associated PTLD was diagnosed. Beside peripheral lymphomas and B symptoms the patient showed neurological symptoms. Examination of the cerebrospinal fluid (CSF) revealed a meningeosis lymphoblastica caused by the EBV lymphoma. Treatment with Rituximab and the antiviral drug Cidofovir led to complete remission with regression of the peripheral lymphomas and disappearance of the neurological symptoms. In addition, the PCR control on EBV DNA became negative in the plasma as well as in CSF. Conclusion: The combination of Rituximab and Cidofovir appears as an interesting alternative treatment in patients with EBV-associated PTLD and CNS involvement. / Hintergrund: Die Epstein-Barr-Virus(EBV)-assoziierte Posttransplantations-lymphoproliferative Disease (PTLD) ist eine gefürchtete Komplikation nach allogener hämatopoetischer Stammzelltransplantation (HSCT). Insbesondere bei Befall des zentralen Nervensystems (ZNS) ist die Behandlung auf Grund der unsicheren Liquorwirksamkeit der meisten Chemotherapeutika als auch von EBV-spezifischen zytotoxischen T-Spenderzellen schwierig. Der monoklonale Anti-CD20-Antikörper Rituximab wurde in den letzten Jahren bei Patienten mit EBV-PTLD intensiv untersucht. Allerdings wurde bislang lediglich von 8 Patienten mit ZNS-Befall eines B-Zell-Lymphoms berichtet, bei denen eine Therapie mit Rituximab erfolgte. Kasuistik: Eine 24-jährige Patientin hatte wegen einer akuten T-lymphoblastischen Leukämie in zweiter kompletter Remission eine allogen-unverwandte, T-Zelldepletierte HSCT erhalten. 10 Monate später wurde eine EBV-assoziierte PTLD diagnostiziert. Neben peripheren Lymphomen und B-Symptomen zeigte die Patientin neurologische Symptome. Die Liquoruntersuchung erbrachte den Befund einer Meningeosis lymphoblastica im Rahmen des EBV-Lymphoms. Die Behandlung mit Rituximab und dem Virustatikum Cidofovir führte zu einer kompletten Remission mit Rückbildung der peripheren Lymphome und Verschwinden der neurologischen Symptomatik. Außerdem wurde die PCR-Kontrolle auf EBV-DNA sowohl im Plasma als auch im Liquor negativ. Schlussfolgerung: Die Kombination von Rituximab und Cidofovir erscheint als eine interessante Therapiealternative für Patienten mit EBV-assoziierter PTLD und ZNS-Befall. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.