1,721,064 research outputs found
Preclinical studies of a new anti-psma (prostate specific membrane antigen) antibody for diagnosis and immunotargeting
No full textIl Prostate Specific Membrane Antigen (PSMA) rappresenta uno dei più promettenti biomarcatori
del carcinoma prostatico. E’ infatti un antigene espresso nell’epitelio prostatico normale ma
iperespresso in un’elevata percentuale di carcinomi prostatici sia organo confinati che metastatici.
Ciò che inoltre, rispetto ad altri antigeni anche di più recente identificazione, rende il PSMA un
target molecolare particolarmente interessante, sia a fini diagnostici che terapeutici, risulta essere la
sua distribuzione a livello della neovascolatura di tumori a vario istotipo.
Il presente lavoro si prefigge di caratterizzare le proprietà di riconoscimento di un nuovo anticorpo
anti-PSMA ottenuto mediante tecnologia degli ibridomi, del rispettivo frammento scFv (single
chain fragment) ottenuto con la tecnologia del DNA ricombinante ed inoltre di analizzare un loro
potenziale utilizzo terapeutico e diagnostico.
Anticorpo anti-PSMA D2/B e scFv D2/B
L’anticorpo anti-PSMA D2/B ha dimostrato, in analisi condotte mediante citofluorimetria, una
notevole specificità di riconoscimento, essendosi dimostrato in grado di riconoscere cellule LNCaP
PSMA+ (MFI=8,538) e di non riconoscere, di contro, cellule PSMA- (DU145, Jurkat, SW780,
MCF-7, CHO). La specificità di riconoscimento in vitro è paragonabile all’anticorpo J591,
attualmente in uso in studi clinici. L’anticorpo D2/B si è dimostrato in grado di spiazzare il legame
di J591 all’antigene, condividendo pertanto con esso un analogo/limitrofo epitopo; inoltre l’affinità
dei due anticorpi è del tutto similare (5nM e 10nM per D2/B e J591, rispettivamente). Il frammento
scFv dell’Mab D2/B, ottenuto mediante tecnologia del DNA ricombinante, dimostra di preservare la
specificità di riconoscimento dell’Ab parentale; [riconoscimento di cellule LNCaP con MFI=2495 e
di cellule PC3, PSMA-, con MFI=210]. L’affinità del scFv si è dimostrata, prevedibilmente, di circa
20 volte inferiore a quella dell’anticorpo parentale, in ragione del sito di legame monovalente; se
resa dimerica tuttavia tale porzione anticorpale differisce, in termini di affinità rispetto all’anticorpo
parentale, di sole 5 volte.
Immunotossine anti-PSMA
Applicando la tecnologia del DNA ricombinante abbiamo clonato la porzione scFv dell’anticorpo
D2/B all’interno del vettore per l’espressione in procarioti pET11d contenente la subunità catalitica
e quella di traslocazione trans-membrana dell’esotossina A di Pseudomonas aeruginosa (PE40). La
proteina di fusione prodotta (scFv-PE40) si è dimostrata in grado di inibire del 50% la
proliferazione (IC50) di una popolazione di cellule LNCaP PSMA+ ad una concentrazione di circa
2*10-11M/L, paragonabile a quella della immunotossina (IT) chimica J591-RTA, da noi
precedentemente studiata e rilevatasi ad elevata potenza citotossica su cellule PSMA+(IC50=3.5*0-11
M/L). Unitamente ad un’elevata efficacia tale immunotossina si è dimostrata capace di un
altrettanto buona specificità; non sono stati infatti raggiunti valori di IC50 su cellule PC3, PSMAnegative,
nemmeno ad una concentrazione di 10-7 M/L. Ad ulteriore conferma di specificità tale
immunotossina ricombinante si è dimostrata citotossica nei confronti della linea cellulare MCF-7
trasfettata con PSMA, se paragonata alla stessa linea trasfettata con il vettore di controllo (IC50
inferiore a 10-11 M/L vs 3*10-8 M/L). Si è infine osservato che l’attività citotossica di una
concentrazione pari a 10-9 M/L dell’immunotossina scFv-PE40 è completamente neutralizzata dalla
contemporanea aggiunta, nel terreno di coltura, di anticorpo anti-PSMA ad una concentrazione di
0.7*10-5M/L.
La IT scFv D2/B-PE40 ha inoltre dimostrato di non essere inattivata nella sua efficacia citocida
dalla presenza di siero umano a diverse concentrazioni derivato sia di pazienti affetti da neoplasia
prostatica che da individui sani, evidenziando, compatibilmente ai limiti di un saggio in vitro, una
notevole stabilità.
Nanoparticelle di oro per imaging dei tessuti neoplastici
Le nanoparticelle (NP) rappresentano per le loro peculiari caratteristiche (i.e. dimensioni dell’ordine
di 10-100 nm, accumulo a livello delle lesioni neoplastiche) un versatile strumento studiato negli
ultimi anni per applicazioni nel campo biomedico. NP coniugate con anticorpi monoclonali o loro
frammenti (i.e. scFv) possono essere utilizzate, in ragione della secificità loro conferita dagli
anticorpi ad esse legati, quali veicoli di molecole tracers o di sostanze terapeutiche. Abbiamo
percio’ deciso di sviluppare, per un possibile utilizzo nell’imaging mediante spettroscopia Raman,
nanoparticelle di oro (AuNP) coniugate all’anticorpo D2/B anti-PSMA e caricate con Texas Red, un
SERS (surface-enhanced Raman scattering) reporter che permette di generare un segnale Raman di
buona intensità.
Le AuNP-D2/B dimostrano in citofluorimetria di legare selettivamente cellule LNCaP PSMA+,
senza di contro generare alcun segnale su cellule PSMA negative. La specificità di legame su
cellule PSMA+ è stata confermata dallo spiazzamento delle AuNP-D2/B mediante l’utilizzo di
concentrazioni crescenti dell’anticorpo D2/B. Abbiamo inoltre dimostrato, mediante microscopia
confocale, l’internalizzazione delle AuNP-D2/B in cellule PSMA+ dopo incubazione a 37°C; in
cellule PSMA negative non è stata di contro rilevata alcuna internalizzazione. La possibilità di
rilevare, mediante spettroscopia Raman, cellule tumorali precedentemente incubate con AuNP è
stata dimostrata “in vitro” utilizzando colture cellulari PSMA+ e PSMA-; cellule LNCaP incubate a
37°C con AuNP-D2/B mostrano un forte segnale Raman. La stessa misura eseguita su cellule
PSMA- non consente di rilevare alcun segnale.
Abbiamo ora intenzione di valutare la biocompatibilità “in vitro” ed “ in vivo” di questo reagente ed
esplorarne le potenzialità nella diagnostica oncologica in modelli murini di carcinoma prostatico.
Validazione in IHC della funzionalità di un Ab anti-hPSMA
L’anticorpo anti-PSMA D2/B si è rivelato in grado, similmente all’anticorpo commerciale 7E11C,
di riconoscere specificamente l’antigene su sezioni di neoplasia prostatica sia fissata in formalina
che congelata. Ulteriori studi sono attualmente in corso per valutare l’efficacia di riconoscimento,
da parte del nostro anticorpo, nei confronti dell’antigene espresso sui neovasi tumorali, la cui
espressione, limitatamente alle neoplasie epiteliali ovariche, è stata da noi confermata con
l’anticorpo commerciale 7E11C.
Modelli Murini PSMA positivi :
Tra le applicazioni di maggior interesse nell’ambito dell’utilizzo clinico delle ITs vi è il trattamento
della malattia micro-/oligometastatica; tra i possibili scenari clinici vi sono pertanto: il trattamento
adiuvante dopo un trattamento con intento di radicalità in casi selezionati ad alto rischio di recidiva
e il trattamento della recidiva biochimica. E’ per tale motivo indispensabile poter disporre di un
modello murino metastatico esprimente l’antigene PSMA. Abbiamo perciò trasfettato la linea
cellulare di melanoma murino B16 con cDNA PSMA-specifico. I trasfettanti hanno dimostrato in
citofluorimetria una notevole espressione di PSMA, a livello di membrana, (MFI=11,578 contro un
MFI=188 del controllo negativo) e si sono inoltre dimostrati sensibili all’effetto citotossico
specifico dell’ IT anti-PSMA scFv D2/B-PE40 (IC50=4*10-9 M/L mentre la IC50 su cellule B16 WT
è >3*10-7 M/L).
Le cellule B16-hPSMA sono state iniettate i.v. in topi C57/BL6 in quantità scalari (0,5, 1 e 2*106);
dopo 21 giorni sono stati analizzati i polmoni degli animali, quali sedi preferenziali di
metastatizzazione. Il numero di metastasi rilevato è stato di circa 140 per gli animali iniettati con 1 e
2*106 cellule e di circa 60 per quelli iniettati con 0,5*106 cellule; mediante western blot si è
dimostrata l’espressione di PSMA nelle metastasi polmonari espiantate dagli animali...Prostate cancer (PCa) is the second leading cause of cancer death in men in western countries.
Whereas primary organ-confined disease can be properly treated and cured by surgery and/or
radiation therapy, there are limited therapeutic options for the advanced forms of this disease
(metastatic, hormone-refractory). Since the conventional therapeutic approaches are poorly
effective in the advanced stages of PCa, several investigations, prompted by the discovery of
antigens expressed in organ-specific patterns, have proposed and studied different immunological
strategies for immunotherapy and immunodiagnosis. Prostate Specific Membrane Antigen (PSMA)
has recently emerged as one of the most promising biomarkers in the diagnosis and treatment of
prostate cancer and its clinical relevance is presently being evaluated in several immunotherapy
trials.
Aims of my work were: produce and characterize a new mAb anti-PSMA and its scFv format and
study their immunotherapeutical and diagnostic applications.
Antibody, scFv fragment and Immunotoxins
Our anti-PSMA mAb D2/B as whole molecule or in scFv format recognizes PSMA expressed on
LNCaP cells with good specificity and affinity; D2/B shows either much affinity than J591 Ab
which is currently under phase II clinical trials; at the same time the scFv format of D2/B shows
only 20-fold lower affinity in respect to whole Ab. Moreover we have demonstrated that our Ab is
able to recognize the PSMA antigen in the native and denaturated form (western Blot, IHC).
When compared to the 7E11c, D2/B mAb shows, in IHC analysis, the same pattern of recognition
both on criopreserved and on formalin fixed prostate cancer tissues; no labeling was demonstrated
on antigen negative normal tissues.
D2/B mAb was chemically linked to ricin A chain toxin obtaining a powerful immunodelivered
drug (immunotoxin, IT) with specific cytotoxic activity on PSMA+ cells; in a cytotoxic assay on
LNCaP cells D2B-RTA IT shows an IC50=10-10 M/L only 35 times higher than the IC50 of J591-
RTA IT (3.5*10-11). In order to reduce the molecular mass of this IT and subsequently improve the
penetration in the tumor mass, we decided to clone the single-chain (scFv) variable region of
PSMA-specific antibody into a prokaryotic expression vector containing the catalytic subunit of
Pseudomonas aeruginosa exotoxin A (PE40). In an 3H-Tdr incorporation assay the PSMA-specific
fusion immunotoxin (IT) inhibit half the proliferation of the PSMA-positive population tested
LNCaP at an estimated concentration of approximately 2*10-11 M/L. No IC50 can be defined on
PSMA-negative PC3 cell line, neither once tested at a concentration of 10-7 M/L. Similarly our IT
shows a great cytotoxicity against hPSMA-transfected MCF-7 cell line (IC50 below 10-11 M/L) but
not against mock-transfected one (IC50 3*10-8 M/L). As a further proof of specificity we observed
that the cytotoxic activity of 10-9 M/L of the abovementioned IT, on LNCaP cells, is fully prevented
by addition of whole molecule PSMA-specific antibody at a concentration of 0,7*10-5 M/L.
Gold Nanoparticles
Specific targeted delivery and control drug release are desirable properties of a drug for tumor
therapy. Nanomedicine, the science which 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 of linked/adsorbed molecules (gene, drug).
We have therefore investigated the binding properties of gold-NPs (20 um Æ) charged with a
reporter solution (Texas red) and conjugated to mAb D2/B. 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. Our results of specific delivery and internalization support our idea to use
mAb anti-PSMA targeted NPs for imaging of tumor sites and to carry toxic drugs inside the tumor.
Syngeneic mouse model:
A syngeneic mouse models has been created transfecting B16 murine melanoma cells with
phCMV3-hPSMA vector. In FACS analysis B16 hPSMA MFI value was 11,578 respect a negative
control value of 188; cytotoxic assays performed on this clone with scFvD2B-PE40 IT show an
IC50= 4*10-9 M/L; the whole toxin PE shows in the same assays an IC50= 6*10-9 M/L. When B16
WT cells have been used the IC50 values were >3*10-7 M/L and 6*10-9 M/L for scFvD2B-PE40 and
PE respectively. To verify the tumorigenicity of our B16-hPSMA in C57BL/6 mice, different
amount of transfected cells 0.5, 1 and 2 millions has been inoculated iv; after 21 days we have
measured the number of metastasis at the lung level. There are about 140 metastasis/lung in mice
inoculated with 1 and 2 millions of cells and 60 metastasis in mice inoculated with 0.5 millions of
cells.
Immunoimaging
The first series of experiments to conjugate Ab D2B with quantum dot, don’t have showed good
results. So we have decided to use the fluorocrome Cy5.5; after conjugation with the fluorocrome
D2/B mAb Ab preserves its ability to recognize LNCaP cells without altering the specificity and
affinity. Therefore we have plan analyze if it was possible to capture “in vivo” the fluorescence
signal of labelled LNCaP cells; we have labeled LNCaP cells “in vitro” with Ab D2B-Cy5.5 and
than we have inoculated SCID mice sc with 1*106 and 7.5*106 cells. With this experiment we have
demonstrated that Explore Optix instrument is able “in vivo” to show the fluorescence signal
derived from the tumor labeled cells.
At the same time we are able to identify “in vivo” the tumor mass, stable tumor obtained
inoculating sc LNCaP cells, injecting iv Ab D2/B labeled with Cy5.5
Anticorpo monoclonale isolato o suo frammento legante l'antigene specifico di membrana della prostata, suoi coniugati e suoi usi
No full textAnticorpo monoclonale isolato o suo frammento legante l'antigene specifico di membrana della prostata (Prostate Specific Membrane Aantigen), preferibilmente nella sua forma nativa presente sulla superficie di cellule tumorali. Vengono forniti inoltre un coniugato dell'anticorpo con un principio attivo e forme modificate del frammento anticorpale legante l'antigene. L'anticorpo intero ed il suo frammento riconoscente l'antigene sono utilizzati da soli o coniugati per il trattamento o la diagnosi di tumori o tessuti associati al tumore overesprimenti PSMA, con preferenza per le neoplasie prostatiche
Isolated monoclonal antibody or fragment thereof binding prostate specific membrane antigen, conjugates and uses thereof
No full textAn isolated monoclonal antibody or fragment thereof binding prostate specific membrane antigen (PSMA) preferably in its native form on the surface of tumour cells. A conjugate of the antibody with an active ingredient and modified forms of the antigen-binding antibody fragment are also provided. The complete antibody and the antigen- recognising fragment thereof are used alone or conjugated for the treatment and the diagnosis of tumours or tissues associated to the tumour overexpressing the PSMA antigen, preferably prostatic neoplastic diseases
Suppression of mTOR pathway in solid tumors: lessons learned from clinical experience in renal cell carcinoma and neuroendocrine tumors and new perspectives
No full textThe PI3K-AKT-mTOR pathway plays role in the regulation of many cellular processes. Hyperactivation of mTOR signaling has been implicated in human carcinogenesis, representing an attractive target for cancer therapy. Among other cancer subtypes, renal cell carcinoma (RCC) and neuroendocrine tumors are relevant settings in which the deregulation of mTOR pathway is of crucial importance. Different mTOR-inhibitory agents have been developed in recent years. Temsirolimus is approved for advanced RCC; everolimus is registered for the treatment of advanced RCC, pancreatic neuroendocrine tumors and postmenopausal, hormone receptor-positive/HER2-negative, advanced breast cancer. This review is focused on the description of the clinical experience with mTOR-inhibitor agents for the treatment of advanced RCC and neuroendocrine tumors, followed by an excursus on the landscape of the ongoing research in this field
Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing.
No full textPathological complete response in a patient affected by multiple synchronous, breast and lung primary malignancies: a case report and review of the literature
No full textA pathological complete response in a patient affected by multiple synchronous, breast and lung primary malignancies is reported. A 63-year-old woman presented with an invasive ductal carcinoma of the breast and a lung adenocarcinoma. After multidisciplinary discussion, the patient underwent pulmonary left lower lobectomy followed by radio-chemotherapy with cisplatin and vinorelbine and started hormone therapy with letrozole. Ten months later, a left mastectomy with axillary lymph nodes dissection was performed. Histologically, a pathological complete response (pCR) was documented. With a review of the Literature, we discuss the issue of multiple primary malignancies, with its diagnostic and therapeutic implications. In cases of multiple synchronous malignancies it has been highlighted the importance of the choice of the best therapeutic approach for both the malignancies, reducing collateral individual effects
The prostate specific membrane antigen regulates the expression of IL-6 and CCL-5 in prostate tumour cells by activating the MAPK pathways
No full textIL-6 and CCL5 are implicated in the development and progression of several forms of tumours incliding PCa. The PSMA expression is augmented in high-grade and metastatic tumours. Some clinical observations suggest that the increased secretion of IL-6 and CCL5 and the higher PSMA expression may be correlated
EpCAM a possible target for immunotoxin treatments of PSMA positive and negative prostate cancer cells
No full textBackground
Prostate carcinoma is the second leading cause of cancer related death in men in western contries; when the disease spreads outside the organ conventional therapies (radio, surgical and chemotherapy) are only palliative. In order to better control the disease progression, in the last then years scientists have discovered and validated various TAAs (tumor associated antigens), as targets of immunological treatments. In a previous study we have demonstrated that PSMA (Prostate Specific Membrane Antigen) can be a good target for immunotoxin therapy of prostate cancer cells; unfortunatly due to the tumor heterogeneity about 43% of the disseminated tumor cells in PCa patients do not show this marker/target.
In this work we have verified the feasibility of using EpCAM (Epithelial Cell Adhesion Molecule) as a second target for a future multitarget approach in prostate cancer.
Results
As first step we have analyzed by flow cytometry the different expression levels of EpCAM antigen on LNCaP, DU145 and PC-3 cells. Using the mAb MOC31recognizing the Ag on the three cell lines, we have confirmed that LNCaP cells express the highest level of Ag and that PC-3 cells show the lowest expression level; the Ab specificity has been assayed on lymphoma Jurkat cells, where the MFI value is superimposable to the negative control MFI value. These analyses show that the PSMA negative DU145 cells expresses a good level of EpCAM antigen. This cells are insensitive to treatment with anti-PSMA IT, because they do not express PSMA Ag. When MOC31 Ab is conjugated to the RTA (Ricin A chain) toxin, the synthesized IT shows high cytotoxic efficacy against the LNCaP and DU145 cells; the activity on PC-3 cells is very low. MOC31-RTA IT is only 8-fold less toxic on DU145 cell than OKT9-RTA IT, a potent but not highly selective anti-TfnR immunotoxin.
MOC31-RTA is effective not only on DU145 cells growing as a monolayer, but it also sterilizes DU145 spheroids (100-150um in diameter), an in vitro model of tumor micrometastasis
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
- …
