Il sistema del complemento come strumento terapeutico nella terapia dei tumori.

2006/2007Il cancro e le malattie cardiovascolari rappresentano le prime cause di morte nei paesi sviluppati; se queste ultime possono beneficiare di terapie chirurgiche e farmacologiche sempre più efficaci, i tumori, soprattutto alcuni tipi, continuano a rappresentare patologie con poche alternative terapeutiche. Nella maggior parte dei casi la chirurgia rappresenta la principale arma per eliminare le masse tumorali. La chemioterapia e la radioterapia cercano soprattutto di eliminare le cellule residue agendo sulla loro continua proliferazione, ma mancano di una reale specificità d’azione e causano ancora notevoli effetti collaterali. Le terapie più innovative puntano invece a sfruttare il sistema immunitario umano come meccanismo effettore. I tumori però derivano da cellule del nostro organismo ed il fatto che abbiano potuto svilupparsi dimostra che il nostro sistema immunitario non li riconosce come estranei e quindi non costruisce contro di essi un’efficiente risposta. I meccanismi effettori del sistema immunitario sono quindi potenzialmente in grado di distruggere in maniera selettiva le cellule tumorali e di causare in questo modo pochissimi effetti collaterali, ma necessitano di qualcosa che indirizzi e faccia partire la loro azione. L’utilizzo degli anticorpi monoclonali come strumento terapeutico vuole appunto sfruttare la loro capacità di raggiungere in maniera molto selettiva uno specifico bersaglio e di attivare di seguito il sistema immunitario. I meccanismi d’azione utilizzati dagli Ab, una volta legati alle cellule tumorali, si basano sull’induzione di processi apoptotici (in maniera dipendente dall’antigene a cui si sono legati), sull’attivazione della citotossicità cellulare e sull’attivazione della cascata complementare. Diversi autori hanno messo in risalto il fatto che gli anticorpi più efficaci in clinica sono proprio quelli con la maggior capacità di sfruttare quest’ultimo meccanismo. A differenza dell’apoptosi e della citotossicità cellulare, il complemento si basa su un sistema di proteine extracellulari che si attivano a cascata e portano alla morte della cellula tumorale, principalmente creando un poro transmembranario e provocando quindi la lisi osmotica del bersaglio. Il suo principale vantaggio sta proprio nell’azione rapida e diretta, che non necessita dell’attivazione di processi intracellulari (apoptosi) o del richiamo di cellule effettrici nella sede in cui si è sviluppato il tumore (citotossicità cellulare). Come detto però, non tutti gli anticorpi si sono dimostrati in grado di attivare la cascata complementare (1); altri, pur attivandola, non portano alla lisi della cellula bersaglio (2); altri ancora, pur avendo un buon effetto citotossico su cellule tumorali in vitro, si sono dimostrati poco efficaci una volta testati in vivo (3). In questi tre anni abbiamo voluto studiare le cause di questi insuccessi e abbiamo cercato di intervenire per proporre delle nuove strategie da utilizzare nell’immunoterapia dei tumori. 1. L’incapacità di attivare la cascata complementare, nella maggior parte dei casi, è da imputare ad un’insufficiente concentrazione degli antigeni associati al tumore; questo non permette di ottenere una congrua vicinanza tra gli anticorpi ad essi legati, che è la condizione essenziale perché attivino il sistema complementare. Utilizzando diversi anticorpi diretti contro epitopi distinti dello stesso antigene associato al tumore è stato possibile dimostrare che è possibile creare una densità anticorpale sufficiente ad ottenere buona attivazione complementare anche sfruttando molecole poco espresse sulla superficie delle cellule tumorali. Un effetto analogo si può ottenere anche utilizzando anticorpi diretti contro due diversi antigeni presenti sulla superficie delle cellule tumorali. 2. Le cellule tumorali derivano da cellule del nostro organismo e come tali esprimono sulla loro superficie delle molecole la cui funzione fisiologica è quella di inibire un’attivazione indesiderata della cascata complementare. Ne deriva che l’espressione, e spesso l’iper-espressione, degli inibitori di membrana del complemento sulle cellule tumorali riduce l’azione degli anticorpi e quindi la lisi delle cellule bersaglio. Sulla base di questo concetto abbiamo pensato di estendere la terapia anticorpale anche a queste molecole, isolando e caratterizzando degli anticorpi in grado di bloccare l’azione degli inibitori di membrana, affiancandoli poi ai comuni anticorpi terapeutici. In questo modo il sistema del complemento, attivato sulle cellule tumorali, non troverebbe ostacoli nella sua azione litica e potrebbe eliminare un maggior numero di cellule tumorali. 3. Se è ipotizzabile un’efficiente azione del complemento su cellule tumorali isolate o aggregati cellulari, è più difficile immaginare la riduzione di una massa tumorale già sviluppata solo attraverso la sua azione. Va ricordato che, se gli anticorpi possono diffondere nell’organismo, non è stata dimostrata la presenza di tutti gli elementi del sistema complementare nel micro-ambiente tumorale. E’ possibile quindi che anticorpi, con ottime prospettive dopo gli esperimenti su cellule in coltura, non dimostrino un reale effetto terapeutico negli esperimenti in vivo semplicemente perché manca il meccanismo effettore nella sede tumorale. Molte cellule dei distretti periferici, e le stesse cellule tumorali, possono produrre alcune proteine della cascata complementare, ma è indubbio che la maggior parte delle molecole del sistema del complemento sono prodotte dal fegato e circolano nel sangue. Creare dei danni ai vasi sanguigni intra-tumorali, oltre ad un effetto “anti-angiogenetico” diretto, permetterebbe quindi il passaggio delle proteine complementari dal circolo al micro-ambiente tumorale ed una migliore azione citotossica degli anticorpi antitumorali. A questo scopo abbiamo focalizzato la nostra attenzione sull’utilizzo di cellule endoteliali umane modificate geneticamente che possono venir reclutate dai vasi in via di formazione nelle masse tumorali ed in seguito venir lisate dal sistema del complemento in maniera specifica ed efficacie; in questo caso si indurrebbe, come auspicato, un aumento della permeabilità vascolare nelle regioni tumorali, con un passaggio di anticorpi anti-tumorali, proteine complementari e cellule effettrici proprio in quella sede. Con questi obiettivi ci siamo concentrati su alcuni modelli sperimentali: a) in primo luogo uno studio sul carcinoma ovarico utilizzando due anticorpi diretti contro due epitopi diversi di un antigene associato a questo tumore e che in precedenza, utilizzati singolarmente, non si erano dimostrati capaci di attivare il complemento. Questo studio è il risultato della collaborazione con l’Istituto Tumori di Milano ed in particolare con il gruppo della dottoressa Silvana Canevari, la quale ha potuto fornirci molti campioni prelevati da pazienti con carcinoma ovarico, utili per confermare i risultati ottenuti su linee cellulari. b) a fianco a questo ci siamo occupati delle leucemie linfatiche croniche, che esprimono sulla superficie cellulare due marker tumorali, CD20 e CD52, e contro cui sono diretti due anticorpi monoclonali utilizzati in clinica, Rituximab e Campath-1H. Sono state utilizzate cellule di pazienti con questa patologia, isolate e caratterizzate dal gruppo del dottor Valter Gattei del Centro di Riferimento Oncologico di Aviano (PN). c) Il Rituximab, primo anticorpo entrato in clinica nella terapia dei tumori, esercita la sua azione principalmente in seguito all’attivazione del sistema complementare; tale azione risulta però limitata dalla presenza degli inibitori di membrana del complemento, come già da noi dimostrato in uno studio in collaborazione con il gruppo della dottoressa Josee Golay dell’Istituto Mario Neri di Milano e degli Ospedali Riuniti di Bergamo. Ogni linea di ricerca è partita con lo studio delle rispettive linee cellulari per avvalorare le idee proposte; i dati ottenuti sono stati poi confermati con analisi su campioni prelevati da pazienti o con modelli animali il più possibile rappresentativi della patologia umana. Una parte dei risultati sono stati oggetto delle pubblicazioni incluse, altri sono stati valorizzati dalla concessione di brevetti sulle molecole prodotte, altri ancora fanno parte di progetti tuttora in corso, ma nel loro insieme rappresentano la base di partenza degli studi che andremo a sviluppare in futuro.XX Cicl

New treatment against Burkitt Lymphoma: targeted-nanoparticles carrying antagomiR17

Motivation: Burkitt Lymphoma is one of the most curable cancers thanks to chemotherapy, but this treatment has side effects because it does not only attack tumor cells, but healthy tissue. It has been discovered the overexpression of miRNA17 in Burkitt Lymphoma cells blocking the apoptosis mechanism, but miRNA17 can be inhibited by an oligonucleotide, antagomiR17. In this project, a new treatment with chitosan nanoparticles functionalized with rituximab (a monoclonal anti-CD20 antibody) and carrying antagomiR17 has been studied in order to solve the off-target problems of chemotherapy.Methods: In vivo, the immunophenotype of two different Burkitt Lymphoma cell lines, Bjab and Raji, were analyzed by flow cytometry and immunofluorescence. In addition, the internalization of chitosan nanoparticles functionalized with rituximab was tested by flow cytometry and confocal microscopy in both Burkitt Lymphoma cell lines. Also, the therapeutic effect of encapsulated antagomiR17 was checked in Bjab cell line. Ex vivo, the binding of modified nanoparticles was determined by immunofluorescence.Results: The immunophenotype confirmed the high expression of CD20 as surface marker in Burkitt Lymphoma cell lines, which is recognized by rituximab. In addition, the internalization of nanoparticles into cells was confirmed. However, the study of the therapeutic effect of antagomiR17 resulted in toxic chitosan nanoparticles for cells. The charge of nanoparticles was modified by adding different polymers (polyethylene glycol, tripolyphosphate, and pluronic acid F68) in the surface in order to reduce the toxicity of nanoparticles by decreasing their positive charge, and studies of binding were made in healthy kidney tissues with these new nanoparticles.Conclusions: Although nanoparticles were internalized into cells, empty chitosan nanoparticles resulted toxic for cells, so modified ones less positive charged were studied in terms of binding. However, more studies are still needed. antagomiR17 must be tested to confirm its therapeutic efficacy in these new active targeted delivery systems

Critical role and therapeutic control of the lectin pathway of complement activation in an abortion-prone mouse mating

The abortion-prone mating combination CBA/J 7 DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J 7 DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J 7 DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss

A comprehensive overview on antibody-drug conjugates: from the conceptualization to cancer therapy

Antibody-Drug Conjugates (ADCs) represent an innovative class of potent anti-cancer compounds that are widely used in the treatment of hematologic malignancies and solid tumors. Unlike conventional chemotherapeutic drug-based therapies, that are mainly associated with modest specificity and therapeutic benefit, the three key components that form an ADC (a monoclonal antibody bound to a cytotoxic drug via a chemical linker moiety) achieve remarkable improvement in terms of targeted killing of cancer cells and, while sparing healthy tissues, a reduction in systemic side effects caused by off-tumor toxicity. Based on their beneficial mechanism of action, 15 ADCs have been approved to date by the market approval by the Food and Drug Administration (FDA), the European Medicines Agency (EMA) and/or other international governmental agencies for use in clinical oncology, and hundreds are undergoing evaluation in the preclinical and clinical phases. Here, our aim is to provide a comprehensive overview of the key features revolving around ADC therapeutic strategy including their structural and targeting properties, mechanism of action, the role of the tumor microenvironment and review the approved ADCs in clinical oncology, providing discussion regarding their toxicity profile, clinical manifestations and use in novel combination therapies. Finally, we briefly review ADCs in other pathological contexts and provide key information regarding ADC manufacturing and analytical characterization

Meniscal ossicles as micro-CT imaging biomarker in a rodent model of antigen-induced arthritis: A synchrotron-based x-ray pilot study

It is increasingly recognized that early detection of bone erosion plays an important role in the overall evaluation of rheumatoid arthritis and in the choice of the correct treatment approach. Since an appropriate use of imaging biomarkers in preclinical settings offers the prospect of smaller and optimized sample size, in the present study we define an anatomical imaging biomarker that could be objectively measured from micro-CT imaging data as an indicator of bone erosion in arthritis process. The well-characterized antigen-induced arthritis (AIA) model in rats was used. The animals were divided into 2 groups: arthritic disease control and arthritic having been administrated with the tumor necrosis factor alpha-blocking agent (Humira). Rats were sacrificed in the acute phase of AIA; peripheral blood and synovial tissue were collected for assessment of arthritis. Ex vivo micro-CT tomography of knee joints was performed at the Elettra synchrotron light source (Trieste, Italy). Overall, results from this study suggest that use of high-resolution micro-CT analysis coupled with meniscal ossicles bone parameters quantification provide a powerful combination to enhance data interpretation and assessment of disease-modifying drugs in an animal model of arthritis

Targeted delivery of neutralizing anti-C5 antibody to renal endothelium prevents complement- dependent tissue damage

Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney

New insight into antiphospholipid syndrome: antibodies to \u3b22glycoprotein I-domain 5 fail to induce thrombi in rats

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of \u3b22glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analysed the in vivo pro-coagulant effect of two groups of 5 serum IgG each reacting selectively with domain 1 or domain 5 in LPS-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy and vascular deposition of \u3b22glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-\u3b22glycoproteinI antibodies served as controls. All the anti-domain 1 positive IgG exhibited potent pro-coagulant activity while the anti-domain 5 positive and the negative control IgG failed to promote blood clot and vessels occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound \u3b22glycoprotein I while able to interact with fluid-phase \u3b22glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit in vivo thrombogenic effect and the interaction of these antibodies with circulating \u3b22glycoproteinI suggest their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining really at risk patients for more appropriate treatments to avoid recurrences and disability

Beta 2 glycoprotein I and neutrophil extracellular traps: Potential bridge between innate and adaptive immunity in anti-phospholipid syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent vascular thrombosis and miscarriages in the absence of known causes. Antibodies against phospholipid-binding proteins (aPL) are pathogenic players in both clotting and pregnancy APS manifestations. There is sound evidence that antibodies specific for beta2 glycoprotein I (β2GPI) trigger thrombotic and pregnancy complications by interacting with the molecule on the membranes of different cell types of the coagulation cascade, and in placenta tissues. In addition to the humoral response against β2GPI, both peripheral and tissue CD4+ β2GPI-specific T cells have been reported in primary APS as well as in systemic lupus erythematosus (SLE)-associated APS. While adaptive immunity plays a clear role in APS, it is still debated whether innate immunity is involved as well. Acute systemic inflammation does not seem to be present in the syndrome, however, there is sound evidence that complement activation is crucial in animal models and can be found also in patients. Furthermore, neutrophil extracellular traps (NETs) have been documented in arterial and venous thrombi with different etiology, including clots in APS models. Keeping in mind that β2GPI is a pleiotropic glycoprotein, acting as scavenger molecule for infectious agents and apoptotic/damaged body constituents and that self-molecules externalized through NETs formation may become immunogenic autoantigens, we demonstrated β2GPI on NETs, and its ability to stimulate CD4+β2GPI-specific T cells. The aim of this review is to elucidate the role of β2GPI in the cross-talk between the innate and adaptive immunity in APS

Invasive meningococcal disease in three siblings with hereditary deficiency of the 8th component of complement: Evidence for the importance of an early diagnosis

Deficiency of the eighth component of complement (C8) is a very rare primary immunodeficiency, associated with invasive, recurrent infections mainly caused by Neisseria species. We report functional and immunochemical C8 deficiency diagnosed in three Albanian siblings who presented with severe meningococcal infections at the age of 15 years, 4 years and 17 months, respectively. The youngest suffered serious complications (necrosis of fingers and toes requiring amputation). METHODS: Functional activity of the classical, alternative and mannose-binding lectin complement pathways was measured in serum from the 3 siblings and their parents (37-year-old woman and 42-year-old man). Forty healthy subjects (20 males and 20 females aged 4-38 years) served as normal controls. Serum complement factors were measured by haemolytic assays and immunoblotting. Sequence DNA analysis of the C8B gene was performed. RESULTS: Analyses of the three complement pathways revealed no haemolytic activity and also absence of C8beta in serum samples from all three siblings. The genetic analysis showed that the three siblings were homozygous for the p.Arg428* mutation in the C8B gene on chromosome 1p32 (MIM 120960). The parents were heterozygous for the mutation and presented normal complement activities. A 2-year follow-up revealed no further infective episodes in the siblings after antibiotic prophylaxis and meningococcal vaccination. CONCLUSIONS: Complement deficiencies are rare and their occurrence is often underestimated. In presence of invasive meningococcal infection, we highlight the importance of complement screening in patients and their relatives in order to discover any genetic defects which would render necessary prophylaxis to prevent recurrent infections and severe complications