Prostate Stem Cell Antigen (PSCA): a putative target for immunotherapy and diagnosis in prostate, pancreatic and bladder carcinoma

L\u2019immunoterapia basata sull\u2019utilizzo di anticorpi non coniugati, coniugati a tossine o radiomarcati, che riconoscono antigeni associati a tumore, \ue8 promettente per la cura di tumori solidi o ematici. Un possibile target per l\u2019immunoterapia potrebbe essere il prostate stem cell antigen (PSCA), un antigene appartenente alla famiglia delle \u201cGPIanchored protein\u201d. Il PSCA \ue8 un antigene di superficie espresso a bassi livelli nel tessuto prostatico sano ed over espresso nel tumore prostatico, pancreatico e della vescica. L\u2019espressione di PSCA \ue8 inoltre correlata positivamente a \u201cGleason score\u201d e allo stadio della patologia nel tumore prostatico. Il presente lavoro di tesi descrive la generazione e caratterizzazione di un anticorpo monoclonale murino anti PSCA (mAb), ottenuta tramite la tecnologia dell\u2019ibridoma, e del suo frammento anticorpale a singola catena (scFv), generato clonando la regione variabile della catena leggera (VL) e della catena pesante (VH) nel vettore di espressione pHEN-2. Tramite citofluorimetria \ue8 stato dimostrato che l\u2019anticorpo monoclinale possiede una buona affinit\ue0 e specificit\ue0 di legame all\u2019antigene. Il potenziale diagnostico dell\u2019anticorpo \ue8 stato dimostrato tramite Western Blot su lisati di tessuti neoplastici di prostrata e pancreas, in cui l\u2019anticorpo \ue8 in grado di legare l\u2019antigene denaturato e glicosilato, e tramite ELISA, in cui l\u2019anticorpo si lega all\u2019antigene espresso da cellule precedentemente fissate. Il potenziale terapeutico dell\u2019anticorpo \ue8 stato valutato tramite saggio di proliferazione: l\u2019anticorpo da solo non \ue8 in grado di indurre morte cellulare tramite un meccanismo diretto, mentre in seguito a coniugazione chimica con la catena A della ricina (RTA) rivela effetto citotossico su cellule PC-3 hPSCA con IC50 (concentrazione in grado di inibire la massima proliferazione cellulare del 50%) pari a 1.3x10-9, valore 100 volte pi\uf9 piccolo di quello ottenuto con la sola tossina RTA. Il frammento scFv \ue8 stato prodotto nel ceppo batterico E. Coli. Mediante analisi citofluorimetrica su cellule PSCA positive e saggio immunoenzimatico sull\u2019antigene ricombinante \ue8 stato verificato che il frammento anticorpale mantiene le stesse caratteristiche di specificit\ue0 di legame all\u2019antigene dell\u2019anticorpo monoclinale parentale, ma possiede affinit\ue0 minore. Quando l\u2019 scFv viene reso bivalente, tramite il cross-linking dei monomeri utilizzando un anticorpo anti-myc, l\u2019affinit\ue0 raggiunge quasi quella dell\u2019anticorpo parentale. Successivamente l\u2019scFv \ue8 stato unito attraverso fusione genetica al dominio enzimatico della tossina batterica Pseudomonas aeruginosa exotoxin A (PE40). L\u2019immunotossina risultante \ue8 espressa nel ceppo batterico E. Coli e si accumula nei corpi d\u2019inclusione. L\u2019analisi citofluorimetrica su cellule PSCA positive fatta utilizzando i corpi d\u2019inclusione rinaturati e contenenti l\u2019immunotossina di fusione ha confermato che l\u2019interazione tra l\u2019scFv e l\u2019antigene viene conservata in seguito alla fusione con la tossina PE40. L\u2019effetto citotossico dell\u2019immunotossina purificata scFv-PE40 verr\ue0 valutata prima in vitro su linee cellulari PSCA positive e negative e poi in modelli in vivo che permetteranno di valutare anche eventuali effetti collaterali.Antibody-based therapy using unconjugated, toxin-conjugated or radiolabeled immunoglobulins recognizing tumor-associated antigens has proven beneficial for solid and hematolymphoid neoplasms. A suitable target could be prostate stem cell antigen (PSCA), a member of the \u201cGPI-anchored protein\u201d. PSCA is a cell surface-antigen expressed at low levels in normal prostate tissue and over expressed in prostate, pancreatic and bladder carcinomas. Moreover PSCA expression is positively correlated with Gleason score and with pathologic stage in prostate cancer. The present thesis describes the generation and characterization of the murine anti PSCA monoclonal antibody (mAb), obtained by hybridoma technology, and its fragment single chain (scFv), generated by cloning the variable heavy (VH) and light (VL) chain sequences in the expression vector pHEN-2. The mAb showed the ability to recognize with good affinity and specificity the native PSCA by flow cytometry. The diagnostic potential of the mAb was demonstrated by Western Blot performed with prostate and pancreatic neoplastic tissue lysates, showing the binding to denaturated and glycosylated PSCA, and by ELISA performed with fixed cells. The mAb was also assessed for its possible use in the therapeutic approach: the cell-proliferation assay demonstrated that the antibody alone is not able to induce cell death through a direct mechanism, while when it is conjugated to the ricin A chain toxin (RTA) by chemical linkage it can poison PC-3 hPSCA cells with an IC50 (i.e. concentration inhibiting 50% of the maximal cell proliferation) of 1.3x10-9 M, value 100 fold lower than the IC50 of the RTA toxin alone. The scFv was produced in E. Coli bacteria; flow cytometric analysis on PSCA-positive cells and immunoenzymatic assay on the recombinant antigen proved that the antibody fragment maintains the binding specificity of the parental monoclonal antibody. The affinity of the scFv is lower than the affinity of mAb but it is partially recovered making the scFv divalent by cross-linking the scFv monomers via an antibody-mediated myc- Tag interaction. To create a fusion immunotoxin (IT) the scFv was later genetically fused to the enzymatic domain of Pseudomonas aeruginosa exotoxin A (PE40). The resulting IT was expressed in E. Coli bacteria and it is accumulated in the inclusion bodies. The flow cytometric analysis on PSCA-positive cells performed with the whole refolded inclusion bodies extract containing the fusion IT confirmed that the interaction of scFv with the PSCA is preserved after fusion to PE40. The efficacy of purified scFv-PE40 will be analyse in vitro on positive and negative cell lines and subsequently in vivo models which also will be useful to study the side effects of this new drug

Auxin and nitric oxide control indeterminate nodule formation

<p>Abstract</p> <p>Background</p> <p>Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using <it>Sinorhizobium meliloti </it>and <it>Rhizobium leguminosarum </it>bacteria genetically engineered for increased auxin synthesis.</p> <p>Results</p> <p>IAA-overproducing <it>S. meliloti </it>increased nodulation in <it>Medicago </it>species, whilst the increased auxin synthesis of <it>R. leguminosarum </it>had no effect on nodulation in <it>Phaseolus vulgaris</it>, a legume bearing determinate nodules. Indeterminate legumes (<it>Medicago </it>species) bearing IAA-overproducing nodules showed an enhanced lateral root development, a process known to be regulated by both IAA and nitric oxide (NO). Higher NO levels were detected in indeterminate nodules of <it>Medicago </it>plants formed by the IAA-overproducing rhizobia. The specific NO scavenger cPTIO markedly reduced nodulation induced by wild type and IAA-overproducing strains.</p> <p>Conclusion</p> <p>The data hereby presented demonstrate that auxin synthesised by rhizobia and nitric oxide positively affect indeterminate nodule formation and, together with the observation of increased expression of an auxin efflux carrier in roots bearing nodules with higher IAA and NO content, support a model of nodule formation that involves auxin transport regulation and NO synthesis.</p

IKKepsilon involvement in Tax-mediated activation of INF pathway

HTLV-1 Tax de-regulates several cellular signaling pathways leading to cell transformation by altering gene expression, intracellular protein distribution and cell proliferation. Tax-1 induces persistent activation of several transcriptional factors and signal transduction pathways, including NF-\u3baB and CREB/ATF. It is known that Tax-1 constitutively activates TAK1 (transforming growth factor-\u3b2-activated kinase 1) and modifies the interferon (INF) regulatory signals by controlling the expression of INF transcription factors 3 (INF3) and INF4. We have recently reported that HTLV-1 and HTLV-2 Tax proteins interact with TAK1-binding protein 2 (TAB2) of the NF-\u3baB pathway and that both Tax proteins transactivate NF-\u3baB promoters [1]. TAB2 functions as an adaptor protein to recruit TAK1 to TRAF2 (TNF-\u3b1 receptor-associated factor) in TNF-\u3b1 signaling pathways. In the present study we have investigated Tax-1 and Tax-2 role in modifying INF and NF-\u3baB activation through the recruitment of IKKepsilon, an I\u3baB kinase homologue involved in NF-\u3baB and INF3 signaling pathways. By co-immunoprecipitation experiments, we have found that both IKKepsilon and Tax-1, but not Tax-2, are present in protein complexes in transfected cells. IKKepsilon and Tax-1 or Tax-2 role in the activation of INF responsive elements or NF-\u3baB containing promoters have been analyzed after transfecting the protein genes in 293T cells and measuring the effect by luciferase assay. Co-expression of Tax-1 and IKKepsilon resulted in an increased IRF activation mediated by IKKepsilon. Interaction of IKKepsilon with Tax-1 and Tax-2 and their possible effects in the de-regulation of the IRF3 pathways will be discussed

Comparison of Tax-1 and Tax-2B post-translational modifications using specific lysine mutants in relation to activation of NF-κB and intracellular localization

ost-translational modifications of HTLV-1 and HTLV-2 Tax-1 and Tax-2 proteins have been shown to play a critical role in their cellular localization, transactivation and protein interactions. Five of ten lysine residues were found to be major targets for Tax-1 modifications: Lys189(K4); Lys197(K5), Lys263(K6), Lys280(K7) and Lys284(K8), are essential for ubiquitination, while sumoylation takes place on Lys280 (K7) and Lys284(K8). Tax-2 contains four additional lysine residues, namely at position Lys100(K2i), Lys149(K3i), Lys185(K3ii), and Lys356(K10i).Very few studies have been so far performed on Tax-2 lysine mutants. We have previously demonstrated that Tax-2B is ubiquitinated and sumoylated similarly to Tax-1. To identify the Tax-2 lysine residues which are directly involved in post-translational modifications, we have constructed a series of Tax-2B mutants with substitutions of lysine (K) residues by arginines (R) and analyzed them for NF-kB and CREB/ATF transactivation, intracellular distribution and extent of ubiquitination and sumoylation. We have found that Tax-2 K7-8R mutant, contrary to its Tax-1 homologue, is only partially affected in its capacity to transactivate NF-\u3baB pathway, is regularly sumoylated and presents formation of nuclear bodies by confocal analysis. However, Tax-2 mutants with extended (K3ii-8R) and/or total (K1-10iR) mutation rate were severely affected for NF-kB transactivation and sumoylation. By comparing Tax-2 WT with mutants K7-8R and K3ii-8R, we observed that the reduction of NF-\u3baB activity is correlated to a parallel decrease in sumoylation. These results suggest that the target for Tax-2 ubiquitination and sumoylation differs from that described for Tax-1

HTLV Tax-1 and Tax-2 proteins enhance interferon regulatory factor 3 dependent promoter activation

The HTLV-1 infection is known to induce an alteration of type I interferon (IFN-I) signaling since it is capable of escaping IFN-mediated immune response in vitro and Tax-1 protein modulates the expression of factors involved in the interferon signaling. In the present study we have investigated the effect of Tax-1 and Tax-2 expression on the activation of an IFN-regulatory factor 3 (IRF3) regulated promoter through the recruitment of the IFN-I upstream IKK\u3b5 and TANK-binding kinase 1 (TBK1) factors, two IkB-related kinase homologues, which are essential for the activation of IRF3 pathway. We have demonstrated that both Tax-1 and Tax-2 were detectable in immuno-complexes formed by IKK\u3b5 and TBK1 in HEK 293T transfected cells, but did not interact with the IRF3 factor. The presence of Tax-1 and IKK\u3b5 in transfected cells resulted in a significant activation of the IRF3 regulated promoter. A similar effect was measured in the presence of Tax-1 and TBK1. We have also observed that Tax-1 mutants defective in sumoylation and ubiquitination post-translational modification were impaired in their ability to form complexes with IKK\u3b5 or TBK1 and in the transactivating activity on IRF3 dependent promoter.These data provide evidence for a role of Tax proteins in the activation of IFN-I pathway, mediated by interaction with IKK\u3b5 and TBK1 kinases. The effects of the Tax interaction with factors that act upstream of interferon regulatory factor IRF3 should be taken into account to further explain the IFN-mediated immune response to HTLV-1 infection. This study is funded by AIRC-Cariverona

Investigation of Properties of Anti-Prostate Specific Membrane Antigen-targeted Gold Nanoparticles as Drug Carriers in Tumor Therapy

Specific targeted delivery and control drug release are desirable properties of a drug for tumor therapy. Nanomedicine, the science that studies the application of nanotechnology to disease treatment, might be of help. Targeted nanoparticles (NP) for their size and structure are able to enhance the accumulation in the tumor of encapsulated or linked / adsorbed molecules (gene, drug). We have therefore investigated the binding properties of gold-NPs (20 nm) conjugated to D2/B, a mAb recognizing the prostate specific membrane antigen (PMSA). PSMA for its wide distribution in prostate tumor (about 70-80% of patients are PSMA+), is an important biomarker in the management of this malignancy using targeted drugs. Gold-NPs have been synthesized by laser ablation, mixed with a reporter solution (Texas red) and treated with a thiol-PEG solution. Derivatization of PEG-COOH with EDC/Sulfo NHS has been used to covalently link the mAb to the NPs. D2/B-NP binding to LNCaP (PSMA+) cells has been assessed by cytometry. We have measured a MFI (mean fluorescence value) of 1,383 for D2/B-NP whereas the MFI of the negative control (CTRL-) was 68. The binding specificity was confirmed on PSMA– Jurkat cells (MFI of 121 and 101 for D2/B-NP and CTRL-, respectively). Binding and internalization of D2/B-NP in LNCaP cells were assayed by confocal microscopy; detection of D2/B-NP by surface-enhanced Raman scattering also revealed the selective binding of the NPs to Ag+ cells. The results of specific delivery and internalization support our idea to use mAb anti-PSMA targeted NPs to enhance the transport of toxic drugs in the tumo