Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL.

Monoclonal antibodies (MAbs) form a central part of chronic lymphocytic leukaemia (CLL) treatment. We therefore evaluated whether complement defects in CLL patients reduced the induction of complement-dependent cytotoxicity (CDC) by using anti-CD20 MAbs rituximab (RTX) and ofatumumab (OFA). Ofatumumab elicited higher CDC levels than RTX in all CLL samples examined, particularly in poor prognosis cohorts (11q- and 17p-). Serum sample analyses revealed that 38.1% of patients were deficient in one or more complement components, correlating with reduced CDC responses. Although a proportion of patients with deficient complement levels initially induced high levels of CDC, on secondary challenge CDC activity in sera was significantly reduced, compared with that in normal human serum (NHS; P<0.01; n=52). In addition, a high CLL cell number contributed to rapid complement exhaustion. Supplementing CLL serum with NHS or individual complement components, particularly C2, restored CDC on secondary challenge to NHS levels (P<0.0001; n=9). In vivo studies revealed that complement components were exhausted in CLL patient sera post RTX treatment, correlating with an inability to elicit CDC. Supplementing MAb treatment with fresh-frozen plasma may therefore maintain CDC levels in CLL patients with a complement deficiency or high white blood cell count. This study has important implications for CLL patients receiving anti-CD20 MAb therapy.

p21((WAF1))-mediated transcriptional targeting of inducible nitric oxide synthase gene therapy sensitizes tumours to fractionated radiotherapy.

Cancer gene therapy that utilizes toxic transgene products requires strict transcriptional targeting to prevent adverse normal tissue effects. We report on the use of a promoter derived from the cyclin dependent kinase inhibitor, p21((WAF1)), to control transgene expression. We demonstrate that this promoter is relatively silent in normal cells (L132, FSK, HMEC-1) compared to the almost constitutive expression obtained in tumour cells (DU145, LNCaP, HT29 and MCF-7) of varying p53 status, a characteristic that will be important in gene therapy protocols. In addition, we found that the p21((WAF1)) promoter could be further induced by both external beam radiation (up to eight-fold in DU145 cells), intracellular-concentrated radionuclides ([(211)At]MABG) (up to 3.5-fold in SK-N-BE(2c) cells) and hypoxia (up to four-fold in DU145 cells). We have previously achieved significant radiosensitization of tumour cells both in vitro and in vivo by using inducible nitric oxide synthase (iNOS) gene therapy to generate the potent radiosensitizer, nitric oxide (NO(.-)). Here, we report that a clinically relevant schedule of p21((WAF1))-driven iNOS gene therapy significantly sensitized both p53 wild-type RIF-1 tumours and p53 mutant HT29 tumours to fractionated radiotherapy. Our data highlight the utility of this p21((WAF1))/iNOS-targeted approach.

Immunoliposomal fenretinide: a novel antitumoral drug for human neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Anti-GD2 monoclonal antibody immunotherapy: a promising strategy in the prevention of neuroblastoma relapse.

In spite of the satisfactory frequency of clinical response to first-line therapy in neuroblastoma (NB), complete eradication of NB cells is rarely achieved. As a consequence, the majority of patients with advanced stage NB undergo relapse, which is often resistant to conventional treatment and rapidly overwhelming. Thus, after induction of the apparent remission, new therapeutic strategies are needed to completely eradicate the small number of surviving NB cells and to prevent relapse. We explored the potential of different doses of the anti-GD2 monoclonal antibody (mAb) 14G2a in an experimental metastatic model where a limited number of HTLA-230 human NB cells are injected i.v. into nude mice, leading to extensive metastases and death of animals within 7-8 weeks. Treatment with 14G2a mAb (1-4 mg/kg cumulative dose given as five i.v. daily administrations) dramatically reduced the metastatic spread of NB cells and prolonged the long-term survival of treated mice in a dose-dependent manner. Neither macrophages nor NK cells appeared to contribute to the protective effect of antibody treatment in vivo, suggesting either an involvement of granulocytes or a complement-mediated cytotoxicity towards NB cells. Whatever the effecting mechanism(s) involved, these results strongly support the clinical use of anti-GD2 mAbs after first-line induction regimens.

Inhibition of neuroblastoma-induced angiogenesis by fenretinide.

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor- (VEGF-) and fibroblast growth factor-2- (FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF- and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48-72 hr of treatment with HPR, followed by a progressive accumulation of cells in G(1) at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.

The function of alkaline phosphatase in the liver: regulation of intrahepatic biliary epithelium secretory activities in the rat.

We studied the effects of alkaline phosphatase (AP) on the secretory processes of the rat intrahepatic biliary epithelium as well as the role of the intrahepatic biliary epithelium in the uptake and biliary secretion of exogenous AP. The effects of acute and chronic administration of AP on bile secretory parameters were investigated in vivo in normal and bile duct ligated (BDL) rats and in vitro in isolated rat bile duct units (IBDU). In vivo, acute AP administration decreased bile flow and biliary bicarbonate excretion and abolished secretin choleresis in BDL rats but not in normal rats. On the contrary, the AP inhibitor, levamisole, increased in BDL rat bile flow and biliary bicarbonate excretion. In vitro, basal and secretin-stimulated Cl(-)/HCO(3)(-) exchanger activity in IBDU was immediately inhibited by AP intraluminal microinjection (apical exposure) but only after a prolonged exposure to the basolateral pole. Levamisole increased the Cl(-)/HCO(3)(-) exchanger activity of IBDU. A significant basolateral uptake of AP occurs in IBDU with a progressive transport to the apical domain. AP chronic treatment increased AP and gamma-glutamyltranspeptidase (gamma-GT) activities in the intrahepatic bile ducts and hepatocyte canalicular pole, promoted enlargement of bile canaliculi, and decreased bile flow and biliary bicarbonate excretion. In conclusion, the intrahepatic biliary epithelium plays a role in the uptake and biliary secretion of serum AP. AP inhibits the secretory processes of the intrahepatic biliary epithelium and induces features of intrahepatic cholestasis after chronic administration. These findings indicate that AP plays an active role in down-regulating the secretory activities of the intrahepatic biliary epithelium.

Effect of pharmacological modulation of liver P-glycoproteins on cyclosporin A biliary excretion and cholestasis: a study in isolated perfused rat liver.

In different cell types P-glycoproteins (P-gp) are involved in the transport of cyclosporin A (CyA). The aim of this study was to evaluate the effect of the pharmacological modulation of the hepatic P-gp on biliary secretion of CyA and on cholestasis induced by acute administration of CyA in the isolated perfused rat liver (IPRL). Verapamil was used as a P-gp specific inhibitor and acetylaminofluorene (AAF) as a P-gp inducer. CyA biliary excretion was determined by administering in the IPRL a tracer dose of [3H]CyA with or without verapamil or AAF. The effect on bile flow was evaluated by administering increasing doses of CyA (2.8, 8, and 20 mg/kg body wt) in the IPRL. Morphological evidence of damage was evaluated by optical and electron microscopy in the liver as well as in primary culture of rat hepatocytes exposed to CyA +/- verapamil. Verapamil significantly inhibited the biliary excretion of a tracer dose of [3H]CyA (0.15+/-0.04 vs 0.33+/-0.07%; P < 0.05). In contrast, pretreatment with AAF significantly increased the biliary excretion of [3H]CyA, (0.61+/-0.10 vs 0.33+/-0.07%; P < 0.05). CyA induced a dose-dependent inhibition of bile flow with a maximal effect at 20 mg/kg CyA (-49.3+/-4.5% decrease of basal bile flow). CyA cholestasis was significantly worsened by the P-gp inhibitor, verapamil (-75.5+/-7.5%; P < 0.05), but it was unaffected by induction of P-gp via AAF pretreatment (-44.9+/-1.7%). During CyA cholestasis, the cumulative biliary excretion of [3H]CyA was lower than in the absence of cholestasis (0.22+/-0.05 vs 0.33+/-0.07%; P < 0.05), was inhibited by verapamil (0.08+/-0.01%; P < 0.05), but was unaffected by AAF (0.23+/-0.05%). No morphological evidence of damage was observed in the liver, and no evidence of cytoskeleton derangement was seen in primary cultures of rat hepatocytes exposed to CyA +/- verapamil. We demonstrated that pharmacological modulation of P-gp may influence the biliary excretion of CyA. The acute cholestatic effect of CyA is worsened by P-gp inhibitors, while it is unaffected by P-gp inducers. This indicates CyA should not be given with other P-gp substrates or inhibitors.