Publications (23)97.79 Total impact
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Article: Redirection of T-cell effector functions for cancer therapy: bispecific antibodies and chimeric antigen receptors.
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ABSTRACT: T cells are the most potent cells of the immune system; however, they fail in the immunosurveillance of tumors. In previous decades, scientists began studying methods to take advantage of T-cell potency in cancer therapy by redirecting them against tumors independently from the T-cell receptor-defined specificity. Among different approaches, the most promising are the use of bispecific antibodies and T-cell engineering to create chimeric antigen receptors. Bispecific antibodies, by simultaneously recognizing target antigen and an activating receptor on the surface of an immune effector cell, offer an opportunity to redirect immune effector cells to kill cancer cells. The other approach is the generation of chimeric antigen receptors by fusing extracellular antibodies to intracellular signaling domains. Chimeric antigen receptor-engineered T cells are able to specifically kill tumor cells in a MHC-independent way. The efficacy of these reagents in different formats has been clinically validated and will be presented here.Future Oncology 04/2013; 9(4):527-39. · 3.16 Impact Factor -
Article: Antitumor effects of a human dimeric antibody fragment 131I-AFRA-DFM5.3 in a mouse model for ovarian cancer.
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ABSTRACT: AFRA-DMF5.3 is a human antibody fragment that, as a dimer, specifically binds to the α-folate receptor (FR) on ovary cancer cells. Pharmacokinetic and biodistribution parameters of (131)I-AFRA-DFM5.3 after intravenous administration in animal models support its potential therapeutic use. We evaluated its preclinical specificity and therapeutic efficacy in tumor models. A negative control, AFRA-DFM6.1, was obtained by protein engineering. The activity and specificity of (131)I-AFRA-DFMs were evaluated by systemic administration (intravenous) in subcutaneous tumor xenograft-bearing nude mice. Pharmacokinetics, biodistribution, and efficacy were assessed by intraperitoneal administration of (131)I-AFRA-DFM5.3 in nude mice bearing 2 different intraperitoneal ovarian carcinoma xenografts. Treatments were tested at different doses and as single or double administrations 1 wk apart. In subcutaneous models, (131)I-AFRA-DFM5.3, but not the negative control, was found to reside on FR-positive tumor masses and significantly reduced tumor growth. In intraperitoneal models, early accumulation on free-floating clumps of ovarian cancer cells and solid peritoneal masses was evident after 1 h, and tumor uptake was stable for up to 3 h. The high tumor uptake determined the efficacy of (131)I-AFRA-DFM5.3. The best antitumor activity, with more than 50% of treated animals cured, was achieved with 2 locoregional treatments of intraperitoneally growing tumors on days 2 and 9. These results suggest that radioimmunotherapy with (131)I-AFRA-DFM5.3 is feasible and leads to significantly prolonged survival. These preclinical data provide the basis for the rationale design of therapeutic treatments of ovarian cancer patients with a radiolabeled anti-FR antibody fragment.Journal of Nuclear Medicine 11/2011; 52(12):1938-46. · 6.38 Impact Factor -
Chapter: Anti-FR Antibody Generation and Engineering: Development of New Therapeutic Tools
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ABSTRACT: The discovery of the methodology to raise mouse monoclonal antibodies (mAbs) represents a milestone in the history of medicine and has opened the way to antibody therapy development. In the oncologic field, antibody-based therapy seems an attractive strategy for those tumors, such as epithelial ovarian cancer and glioblastoma, for which the existing treatment options are still not sufficient. Initial clinical trials with mouse mAbs enlighten, as major limitations, their xenogenic origin and their dimension. Thus, in order to optimize mAb clinical therapeutic applications, genetic engineering was developed to: (1) generate chimeric, humanized, and human mAbs starting from mouse mAbs; (2) reshape antibody format; (3) increase antibody efficacy. The history of anti-human folate receptor (FR)α mAb generation and its modification paralleled that of genetic engineering of mAbs. At least three anti-FRα mAbs (MOv18, MOv19, and LK26) and their derivatives have reached advanced levels of development. In this chapter, the most relevant preclinical and clinical results obtained with them are widely discussed. Also, published data related to anti-FRβ mAb are reported. Full exploitation of the described anti-FRα antibody-based reagents, however, awaits the confirmation of promising drug safety and clinical efficacy from well-designed, randomized clinical trials. KeywordsAntibody–Antibody fragments–Folate receptor–Ovarian cancer–Therapy05/2011: pages 151-179; -
Article: Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.
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ABSTRACT: To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.PLoS ONE 01/2011; 6(7):e22081. · 4.09 Impact Factor -
Article: Shed HER2 extracellular domain in HER2-mediated tumor growth and in trastuzumab susceptibility.
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ABSTRACT: The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy.Journal of Cellular Physiology 10/2010; 225(1):256-65. · 3.87 Impact Factor -
Article: Protective versus pathogenic anti-CD4 immunity: insights from the study of natural resistance to HIV infection.
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ABSTRACT: HIV-1 exposure causes several dramatic unbalances in the immune system homeostasis. Here, we will focus on the paradox whereby CD4 specific autoimmune responses, which are expected to contribute to the catastrophic loss of most part of the T helper lymphocyte subset in infected patients, may display the characteristics of an unconventional protective immunity in individuals naturally resistant to HIV-1 infection. Reference to differences in fine epitope mapping of these two oppositely polarized outcomes will be presented, with particular reference to partially or totally CD4-gp120 complex-specific antibodies. The fine tuning of the anti-self immune response to the HIV-1 receptor may determine whether viral exposure will result in infection or, alternatively, protective immunity.Along this line, an efficacious anti-HIV strategy can rely on the active (i.e., through immunization) or passive targeting of cryptic epitopes of the CD4-gp120 complex, including those harboured within the CD4 molecule. Such epitopes are expected to be safe from genetic drift and thus allow for broad spectrum of efficacy. Moreover, since these epitopes are not routinely exposed in uninfected individuals, they are expected to become targets of neutralizing antibodies or other specifically designed molecules only after viral exposure, with a predictable low impact in terms of potentially harmful anti-CD4 self-reactivity.The experimentum naturae of naturally resistant individuals indicates a strategy to design innovative strategies to neutralize HIV-1 by acting on the sharp edge between harmful and protective self-reactivity.Journal of Translational Medicine 11/2009; 7:101. · 3.41 Impact Factor -
Article: (177)Lu- labeled MOv18 as compared to (131)I- or (90)Y-labeled MOv18 has the better therapeutic effect in eradication of alpha folate receptor-expressing tumor xenografts.
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ABSTRACT: The mouse monoclonal antibody MOv18, directed against the alpha-isoform of folate receptor (FR), was investigated to identify the optimal radioconjugate for radioimmunotherapy of minimal residual disease in ovarian cancer. Pharmacokinetics, biodistribution, long-term therapeutic efficacy and toxicity of MOv18, labeled with the beta-emitters (131)I, (90)Y and (177)Lu, were compared in a xenografted mouse model, composed by two cell lines, A431FR and A431MK, differing only for FR expression. A shorter blood clearance and a higher tumor uptake were observed for (90)Y- and (177)Lu- compared to (131)I-MOv18, and a shorter blood pharmacokinetics was recorded in A431FR-bearing animals. At equitoxic maximum tolerable doses, the general irradiation by (131)I- and (90)Y-MOv18 gives rise to strong targeted effects on A431FR and nontargeted effects on A431MK tumors, while (177)Lu-MOv18 was able to eradicate small size tumor masses expressing the antigen of interest exerting only mild non-targeted effects. (177)Lu-MOv18 at the maximal tolerated dose is the immunoradioconjugate with the best therapeutic window in experimental conditions of small tumor volume.Nuclear Medicine and Biology 11/2009; 36(7):759-70. · 3.02 Impact Factor -
Article: Protective versus pathogenic anti-CD4 immunity: insights from the study of natural resistance to HIV infection
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ABSTRACT: Abstract HIV-1 exposure causes several dramatic unbalances in the immune system homeostasis. Here, we will focus on the paradox whereby CD4 specific autoimmune responses, which are expected to contribute to the catastrophic loss of most part of the T helper lymphocyte subset in infected patients, may display the characteristics of an unconventional protective immunity in individuals naturally resistant to HIV-1 infection. Reference to differences in fine epitope mapping of these two oppositely polarized outcomes will be presented, with particular reference to partially or totally CD4-gp120 complex-specific antibodies. The fine tuning of the anti-self immune response to the HIV-1 receptor may determine whether viral exposure will result in infection or, alternatively, protective immunity. Along this line, an efficacious anti-HIV strategy can rely on the active ( i.e ., through immunization) or passive targeting of cryptic epitopes of the CD4-gp120 complex, including those harboured within the CD4 molecule. Such epitopes are expected to be safe from genetic drift and thus allow for broad spectrum of efficacy. Moreover, since these epitopes are not routinely exposed in uninfected individuals, they are expected to become targets of neutralizing antibodies or other specifically designed molecules only after viral exposure, with a predictable low impact in terms of potentially harmful anti-CD4 self-reactivity. The experimentum naturae of naturally resistant individuals indicates a strategy to design innovative strategies to neutralize HIV-1 by acting on the sharp edge between harmful and protective self-reactivity.Journal of Translational Medicine. 01/2009; -
Article: Activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression.
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ABSTRACT: Prostate cancer remains the second most frequent cause of tumor-related deaths in the Western world. Additional markers for the identification of prostate cancer development and progression are needed. Osteopontin (OPN), which activates matrix metalloproteinases (MMP), is considered a prognostic biomarker in several cancers. "In silico" and experimental approaches were used to determine whether OPN-mediated MMP activation may be a signal of prostate cancer progression. Pearson correlation coefficients were computed for each OPN/MMP pair across seven publicly available prostate cancer gene expression data sets. Using Gene Set Enrichment Analysis, 101 cancer-related gene sets were analyzed for association with OPN and MMP-9 expression. OPN, MMP-9, MMP-2 tissue inhibitor of metalloproteinase-1 plasma levels, and MMP gelatinase activity were measured by ELISA and zymography in 96 and 92 patients with prostate cancer and benign prostatic hyperplasia, respectively, and 125 age-matched healthy men. Computational analyses identified a significant correlation only between MMP-9 and OPN, and showed significant enrichment scores in "cell proliferation", "genes constituting the phosphoinositide-3-kinase predictor", "proliferation signature", and "tumor metastasis" gene sets in association with both OPN and MMP-9. Plasma analyses revealed a significant increase in OPN and MMP-9 levels and activity in patients with prostate cancer in association with clinical variables (prostate-specific antigen > 4 ng/mL and Gleason score > 7). Significant correlation between OPN and MMP-9 levels were also observed. Mean plasma levels of OPN and MMP-9 decreased in patients with prostate cancer within 6 months after prostatectomy. The concordant computational and experimental data indicate that the extent of OPN pathway activation correlates with prostate cancer progression.Clinical Cancer Research 11/2008; 14(22):7470-80. · 7.74 Impact Factor -
Article: Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor.
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ABSTRACT: We previously developed murine and chimeric antibodies against a specific epithelial ovarian carcinoma (EOC) marker, named folate receptor (FR), and promising results were obtained in phase II trials. More recently, we successfully generated a completely human Fab fragment, C4, by conversion of one of the murine anti-FR antibodies to human antibody using phage display and guided selection. However, subsequent efforts to obtain C4 in a dimer format, which seems especially desirable for EOC locoregional treatment, resulted in a highly heterogeneous product upon natural dimerization and in a very poor production yield upon chemical dimerization by a non-hydrolyzable linker to a di-Fab-maleimide (DFM). We therefore designed, constructed and characterized a large Fab dual combinatorial human antibody phage display library obtained from EOC patients and potentially biased toward an anti-tumor response in an effort to obtain new anti-FR human antibodies suitable for therapy. Using this library and guiding the selection on FR-expressing cells with murine/human antibody chains, we generated four new human anti-FR antibody (AFRA) Fab fragments, one of which was genetically and chemically manipulated to obtain a chemical dimer, designated AFRA-DFM5.3, with high yield production and the capability for purification scaled-up to clinical grade. Overall affinity of AFRA-DFM5.3 was in the 2-digit nanomolar range, and immunohistochemistry indicated that the reagent recognized the FR expressed on EOC samples. (131)I-AFRA-DFM5.3 showed high immunoreactivity, in vitro stability and integrity, and specifically accumulated only in FR-expressing tumors in subcutaneous preclinical in vivo models. Overall, our studies demonstrate the successful conversion of murine to completely human anti-FR antibodies through the combined use of antibody phage display libraries biased toward an anti-tumor response, guided selection and chain shuffling, and point to the suitability of AFRA5.3 for future clinical application in ovarian cancer.Cancer Immunology and Immunotherapy 09/2008; 58(4):531-46. · 3.70 Impact Factor -
Article: Dissecting the structural determinants of the interaction between the human cytomegalovirus UL18 protein and the CD85j immune receptor.
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ABSTRACT: UL18 is a glycoprotein encoded by the human cytomegalovirus genome and is thought to play a pivotal role during human cytomegalovirus infection, although its exact function is still a matter of debate. UL18 shares structural similarity with MHC class I and binds the receptor CD85j on immune cells. Besides UL18, CD85j binds MHC class I molecules. The binding properties of CD85j to MHC class I molecules have been thoroughly studied. Conversely, very little information is available on the CD85j/UL18 complex, namely that UL18 binds CD85j through its alpha3 domain with an affinity that is approximately 1000-fold higher than the MHC class I affinity for CD85j. Deeper knowledge of features of the UL18/CD85j complex would help to disclose the function of UL18 when it binds to CD85j. In this study we first demonstrated that the UL18alpha3 domain is not sufficient per se for binding and that beta2-microglobulin is necessary for UL18-CD85j interaction. We then dissected structural determinants of binding UL18 to CD85j. To this end, we constructed a three-dimensional model of the complex. The model was used to design mutants in selected regions of the putative interaction interface, the effects of which were measured on binding. Six regions in both the alpha2 and alpha3 domains and specific amino acids within them were identified that are potentially involved in the UL18-CD85j interaction. The higher affinity of UL18 to CD85j, compared with MHC class I, seems to be due not to additional interaction regions but to an overall better fit of the two molecules.The Journal of Immunology 02/2008; 180(2):957-68. · 5.79 Impact Factor -
Article: Redirected activity of human antitumor chimeric immune receptors is governed by antigen and receptor expression levels and affinity of interaction.
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ABSTRACT: Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non-major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the zeta-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.Journla of Immunotherapy 11/2007; 30(7):684-93. · 3.27 Impact Factor -
Article: The use of a tropism-modified measles virus in folate receptor-targeted virotherapy of ovarian cancer.
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ABSTRACT: Attenuated measles viruses are promising experimental anticancer agents currently being evaluated in a phase I dose escalation trial for ovarian cancer patients. Virus attachment, entry, and subsequent intercellular fusion between infected and uninfected neighboring cells are mediated via the two measles receptors (CD46 and SLAM). To minimize potential toxicity due to measles virus-associated immunosuppression and infection of nontarget tissues, we sought to develop an ovarian cancer exclusive fully retargeted measles virus. Interactions of measles virus with its natural receptors were ablated, and a single-chain antibody (scFv) specific for alpha-folate receptor (FRalpha), a target overexpressed on 90% of nonmucinous ovarian cancer, was genetically engineered on the viral attachment protein (MV-alphaFR). Specificity of virus tropism was tested on tumor and normal cells. Biodistribution of measles virus infection was evaluated in measles-susceptible CD46 transgenic mice, whereas antitumor activity was monitored noninvasively by bioluminescence imaging in xenograft models. Tropism and fusogenic activity of MV-alphaFR was redirected exclusively to FRalpha without compromise to virus infectivity. In contrast to the parental virus, MV-alphaFR has no background infectivity on normal human cells. The antitumor activity of MV-alphaFR, as assessed by tumor volume reduction and overall survival increase, was equal to the parental virus in two models of human ovarian cancer (s.c. and i.p.). A FR-exclusive ovarian cancer targeted oncolytic virus was generated and shown to be therapeutically effective, thus introducing a new modality for FR targeting and a candidate measles virus for clinical testing.Clinical Cancer Research 11/2006; 12(20 Pt 1):6170-8. · 7.74 Impact Factor -
Article: Complement activated by chimeric anti-folate receptor antibodies is an efficient effector system to control ovarian carcinoma.
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ABSTRACT: Two chimeric monoclonal antibodies (mAb), cMOV18 and cMOV19, recognizing distinct epitopes of folate receptor highly expressed on epithelial ovarian cancer (EOC) cells were analyzed for their ability to activate complement (C) as a means to enhance their antitumor activity. The individual cMOVs failed to activate C on six EOC cell lines as documented by the marginal deposition of C components and the negligible C-dependent cytotoxicity (CDC). Conversely, the mixture of cMOVs was more effective, although the percentage of cell killing did not exceed 25%. Fluorescence-activated cell sorting analysis of EOC cells for surface expression of the membrane C regulatory proteins (mCRP) revealed high levels of CD46, variable expression of CD59, and absence of CD55. This finding was confirmed in tumor tissue specimens obtained from advanced-stage EOC patients and analyzed for the expression of mCRPs mRNA using a cDNA microarray and for the presence of proteins by immunohistochemistry. Incubation of EOC cells with neutralizing mAbs to CD46 and CD59 led to a significant increase in the CDC from 10%-20% to 45%-50%. The relative contribution of antibody-dependent cell cytoxicity (ADCC) and C-dependent killing of two EOC cell lines induced by the mixture of cMOV18 and cMOV19 was about 15% and 25%-35%, respectively, bringing the total killing to about 40%-50%. This value increased to 60%-70% after neutralization of CD46 and CD59 without an appreciable change of ADCC. These results suggest that C is the major contributor to the killing of EOC cells induced by the mixture of cMOV18 and cMOV19.Cancer Research 05/2006; 66(7):3876-83. · 7.86 Impact Factor -
Article: 90Y Labeling of monoclonal antibody MOv18 and preclinical validation for radioimmunotherapy of human ovarian carcinomas.
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ABSTRACT: The monoclonal antibody (mAb) MOv18 binds the membrane alpha isoform of the folate receptor (FR) which is overexpressed in human ovarian carcinoma cells. Exploiting the targeting capacity of this mAb, we developed and preclinically validated a protocol for the stable labeling of the mAb with 90Y, an isotope which has shown promise in cancer radioimmunotherapy. MOv18 was derivatized with the stable macrocyclic ligand p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (Bz-DOTA). MOv18-Bz-DOTA conjugates were labeled with 90Y or 111In under metal-free and good laboratory practice conditions. At the optimal Bz-DOTA/mAb derivatization ratio of 4-5, conjugates maintained binding activity up to 6 months, were efficiently labeled with 90Y or 111In (mean labeling yield 85 and 64%, associated to a final mean specific activity of 74 and 37 MBq/mg) and displayed a mean immunoreactivity of 60 and 58%, respectively. The radiolabeled preparations were stable in human serum, with >97% radioactivity associated to mAb at 48 h after labeling. The ability of 90Y- and 111In-MOv18 to localize FR on tumors in vivo was analyzed in nude mice bearing tumors induced by isogenic cell lines differing only in the presence or absence of the relevant antigen [A431FR (FR-positive) and A431tMock (FR-negative)]. In vivo biodistribution in organs other than tumor was comparable in non-tumor-, A431tMock- and A431FR-bearing mice, whereas the median tumor uptake of the radiolabeled reagents, expressed as area under the curve (AUC) and maximum uptake (Umax), was significantly higher (sixfold to sevenfold) in A431FR than in A431tMock tumors (P=0.0465 and P=0.0332, respectively). Mean maximum uptake (% ID/g) for 90Y-MOv18 was 53.7 and 7.4 in A431FR and A431tMock respectively; corresponding values for 111In-Mov18 were 45.0 and 11.3. These data demonstrate the feasibility of 90Y-labeling of MOv18 without compromising antibody binding ability and the immunoreagent-specific localization in vivo on FR-expressing tumors, suggesting the suitability of 90Y-MOv18 for clinical studies.Cancer Immunology and Immunotherapy 12/2005; 54(12):1200-13. · 3.70 Impact Factor -
Article: Highly efficient redirected anti-tumor activity of human lymphocytes transduced with a completely human chimeric immune receptor.
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ABSTRACT: Novel antibody-based immunotherapeutic strategies exploit chimeric immune receptors (CIR), expressed on the surface of transduced human peripheral blood mononuclear cells (PBMC), to redirect potent non-MHC-dependent cytotoxicity to tumor cells expressing a tumor-associated antigen. However, clinical application of the strategy has been hampered by the potential side effects associated with immunogenicity and by low transduction efficiency. A fully human CIR was constructed that triggers immune activation through the zeta chain of CD3 and contains a human single-chain antibody fragment specific for an extracellular epitope of HER2. PBMC were transduced with the CIR using gibbon-ape leukemia virus envelope pseudotyped retroviruses. In vitro cytotoxicity and inhibition assays were carried out using normal and tumor cell lines expressing different levels of HER2. Bulk populations of CIR-transduced PBMC could express high levels of the construct and subcloning ensured stable expression. CIR-mediated killing and growth inhibition of targets expressing high HER2 levels were very efficient at low effector-to-target ratios. Under the same experimental conditions, CIR-mediated activity against normal cells expressing low HER2 levels was marginal. The CIR-mediated recognition of target cells induced the release of soluble factors able to inhibit growth of both HER-positive and HER2-negative bystander tumor cells. Human CIR-transduced PBMC exert a potent and dose-dependent anti-tumor activity. Target antigen level appeared to be a critical determinant of specificity and delivery of signals leading to redirected effector functions. Soluble factors, released by redirected effectors at the site of antigen-driven activation, mediate potent bystander killing.The Journal of Gene Medicine 02/2005; 7(2):158-70. · 2.48 Impact Factor -
Article: A novel isoform of pro-interleukin-18 expressed in ovarian tumors is resistant to caspase-1 and -4 processing.
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ABSTRACT: Interleukin-18 (IL-18) is a proinflammatory cytokine synthesized as a 24 kDa inactive precursor (pro-IL-18) by several cell types, and is processed to a bioactive molecule of 18 kDa by the proteinases caspase-1 or caspase-4. All ovarian carcinoma cell lines express pro-IL-18, only in some instances coexpress caspase-1, and always express caspase-4; in any case, they display a defective processing of IL-18. We analysed whether pro-IL-18, present in two ovarian carcinoma cell lysates, could be processed 'in vitro' by recombinant active caspase-1. While most of pro-IL-18 could be cleaved by caspase-1, a residual of pro-IL-18 appeared to be resistant. Cloning and sequence analysis of the whole pro-IL-18 open reading frame demonstrated the existence of an alternatively spliced mRNA variant, which lacked exon-3 (Delta3pro-IL-18). The 12 bp exon-3 encodes for the AEDD amino-acid sequence, which is N-terminal with respect to the cleavage site of caspase-1. Both pro-IL-18 and Delta3pro-IL-18 mRNA isoforms were detected in all ovarian cancer cell lines analysed, while Delta3pro-IL-18 mRNA was undetectable in normal ovarian epithelial cells. The Delta3pro-IL-18 cDNA induced synthesis of an alternative Delta3pro-IL-18 protein upon transfection into a murine cell line. The Delta3pro-IL-18 protein was resistant to proteolytic activation by caspase-1 and -4, although it was capable to bind caspase-1. Aternative splicing of pro-IL-18 exon-3 may represent a novel mechanism of regulation of bioactive IL-18 production in human ovarian tumors.Oncogene 10/2004; 23(45):7552-60. · 6.37 Impact Factor -
Article: CD95-mediated apoptosis is impaired at receptor level by cellular FLICE-inhibitory protein (long form) in wild-type p53 human ovarian carcinoma.
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ABSTRACT: Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status. CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays. CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIP(L)) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIP(L) down-modulation at the DISC level. Down-regulation of c-FLIP(L) with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIP(L) in apoptosis resistance. Removal of c-FLIP(L) block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIP(L) involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIP(L) expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIP(L) and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status. The inhibitory protein c-FLIP(L) is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.Clinical Cancer Research 09/2004; 10(15):5202-14. · 7.74 Impact Factor -
Article: Isolation of human Fab fragments against ovarian carcinoma using guided selection.
Methods in molecular biology (Clifton, N.J.) 02/2003; 207:145-59. -
Article: Re: Blocking oncogenic Ras signaling for cancer therapy.
JNCI Journal of the National Cancer Institute 08/2002; 94(13):1031-2; author reply 1032. · 13.76 Impact Factor
Top co-authors
Top Journals
Institutions
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2004–2013
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Fondazione IRCCS Istituto Nazionale dei Tumori di Milano
Milano, Lombardy, Italy
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2002–2003
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Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori
Meldola, Emilia-Romagna, Italy
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