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  • 1
    In: Neuropharmacology, Elsevier BV, Vol. 79 ( 2014-04), p. 119-126
    Type of Medium: Online Resource
    ISSN: 0028-3908
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    Language: English
    Publisher: Elsevier BV
    Publication Date: 2014
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  • 2
    In: Biology of Blood and Marrow Transplantation, Elsevier BV, Vol. 16, No. 9 ( 2010-09), p. 1293-1301
    Type of Medium: Online Resource
    ISSN: 1083-8791
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2010
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  • 3
    In: Biology of Blood and Marrow Transplantation, Elsevier BV, Vol. 23, No. 12 ( 2017-12), p. 2070-2078
    Type of Medium: Online Resource
    ISSN: 1083-8791
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2017
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    detail.hit.zdb_id: 2057605-5
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  • 4
    In: Oncotarget, Impact Journals, LLC, Vol. 7, No. 32 ( 2016-08-09), p. 51581-51597
    Type of Medium: Online Resource
    ISSN: 1949-2553
    URL: Issue
    Language: English
    Publisher: Impact Journals, LLC
    Publication Date: 2016
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  • 5
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 2201-2201
    Abstract: Background Acute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed. Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML. Among the surface molecules identified, the CD33 molecule represents so far one of the main validated target in AML and, being broadly expressed on AML blasts, represents a suitable antigen to be targeted with CAR-T cells. Objectives The aim of the present study is to preclinically evaluate the efficacy and safety profiles of CD33.CAR redirected Cytokine Induced Killer (CIK) cells alone and in combination with standard chemotherapeutic agents. Methods Donor derived- and autologous-CIK cells were stably or transiently transduced with a third generation anti-CD33.CAR by Sleeping Beauty transposon- or mRNA-mediated engineering. In vitro anti-AML activity has been assessed by means of Flow cytometry-based cytotoxicity (AnnV-7AAD staining), proliferation (Ki67 staining and CFSE dilution) and cytokine production (intracellular IFNg and IL2 detection) assays, upon challenge with AML samples. In vivo efficacy has been evaluated in NSG mice transplanted with MA9-NRas AML cell line or primary AML samples. Moreover, an already established xenograft chemotherapy model has been exploited to examine the potential benefit of combining CD33.CAR-CIK cells with standard AML induction therapy (Ara-C and doxorubicin). Results CD33.CAR stably expressing CIK cells were able to induce a potent anti-leukemic activity in vitro, in terms of specific killing either in short term ( 〉 70% at 4h, E:T ratio 5:1) and long term cytotoxic assays ( 〉 90% at 1 week, E:T ratio 1:10), with statistically significant differences as compared to the unmanipulated condition. Moreover, CD33.CAR-CIK cells were able to retain a significant cytotoxic activity when re-challenged with the CD33+ target following a previous stimulation (up to 65%). The proliferative response to AML target cells was also considerable and CAR-specific (up to 60% of Ki67+CAR-CIK cells and up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% of IFN-γ producing CAR-CIK cells and up to 25% of IL-2 producing CAR-CIK cells). CIK cells transiently expressing the CD33.CAR were also effective towards the AML target. In vivo results showed that CD33.CAR-CIK cells were able to control the disease in MA9 grafted mice in all the districts analyzed (peripheral blood, bone marrow, spleen, liver and kidney), as compared to untreated mice. To evaluate the effect of CD33.CAR-CIK cell immunotherapy particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an early treatment approach, treating mice 5 days after i.v. injection of a secondary transplanted PDX sample. We observed a clear engraftment reduction in the treated cohort, nearly undetectable in 2 out 5 mice, while a high leukemic burden has been detected in untreated mice (up to 70% of engraftment in bone marrow). Furthermore, by exploiting CD33.CAR-CIK cell treatment in mice experiencing disease recurrence after the "5+3" chemotherapy-induction protocol, preliminary data showed that CD33.CAR-CIK cells were also capable to target chemotherapy resistant/residual AML cells. Conclusions Considering our in vivo preliminary results, we aim to further evaluate CD33.CAR-CIK cell immunotherapy efficacy, particularly against chemotherapy resistant/residual AML cells. Concerning the safety aspect, since the CD33 targeting raises concerns for a potential myelotoxicity, we will assess the potential long-term off-target effects of CD33.CAR-CIK cells (comparing stably with transiently expressed CD33.CARs) on normal hematopoietic stem/myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 6
    Online Resource
    Online Resource
    Ovid Technologies (Wolters Kluwer Health) ; 2015
    In:  Current Opinion in Hematology Vol. 22, No. 6 ( 2015-11), p. 497-502
    In: Current Opinion in Hematology, Ovid Technologies (Wolters Kluwer Health), Vol. 22, No. 6 ( 2015-11), p. 497-502
    Type of Medium: Online Resource
    ISSN: 1065-6251
    Language: English
    Publisher: Ovid Technologies (Wolters Kluwer Health)
    Publication Date: 2015
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  • 7
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 1359-1359
    Abstract: In the last years, adoptive cellular immunotherapy employing T lymphocytes genetically modified with Chimeric Antigen Receptors (CARs) has demonstrated impressive clinical results, particularly in the treatment of acute/chronic lymphoblastic leukemia and B-cell lymphoma, paving the way towards the possibility to translate this approach also to other hematological malignancies, such as Acute Myeloid Leukemia (AML). In the AML context, the CD123 antigen (IL-3 receptor alpha subunit) represents a good target antigen, being a poor prognosis over-expressed marker on AML cells and leukemic stem cells (LSCs), a rare population that plays a key role in perpetuating leukemia. However, CD123 is also expressed on the surface of healthy cells such as monocytes and endothelial cells, although at lower levels as compared to leukemic cells. The potential recognition of low antigen positive healthy tissues by CAR-redirected T cells, through the so called "on-target-off-organ" effect, limits a safe clinical employment of this immunotherapeutic approach. CARs are artificial receptors generated by joining the cytoplasmic TCR (T Cell Receptor) signaling modules to the heavy and light chain variable regions of a monoclonal antibody, whose affinity toward a target antigen is a variable capable of influencing the CAR-mediated functional responses. Therefore, in our study we investigated how the CAR affinity variable in the context of CD123 targeting, together with the CAR and CD123 target antigen density, could impact anti-CD123. CAR-redirected effector cells efficacy against leukemic cells and safety towards the healthy cells. To this aim, Cytokine-Induced Killer (CIK) effector cells have been genetically modified with four Chimeric Affinity Mutants (CAMs), CAM-1, CAM-2, CAM-3 and CAM-4, identified by means of a computational docking technique. In vitro cytotoxic assays, cytokine production and proliferation experiments have been performed in order to evaluate both the efficacy and safety profile of the CAR-redirected CIK cells, using un-manipulated CIK cells (NO DNA) and wild-type anti-CD123.CAR condition as controls. The functional characterization of all the CAMs revealed both the specificity and the effectiveness of CIK-CAR+ cells against the CD123+ THP-1 cell line and primary AML cells. However we observed that, at least in the context of CD123 targeting, a good CAR expression level is necessary for inducing effective later functions, such as proliferation and cytokine production, towards a high CD123+ target. When introducing leukemic cell lines with different CD123 density on their surface we observed different effector properties minimally influenced by the CAR affinity. In particular, in terms of killing activity, we noticed that a number of ≈1600 CD123 molecules is sufficient to induce a good cytotoxic response of all the CARs tested, with the CAM-2 (2-magnitude log lower in affinity) being less powerful. At the same time, this antigen density is not enough to determine a good proliferative capability which instead occurs with leukemic target cells expressing 5000 or more CD123 molecules. Considering the cytokine production (IL-2 and IFN-gamma), we observed that all CIK-CAR+ cells showed a cytokine release that is directly proportional to the target antigen density, with CAM-2 showing a reduced response towards low-CD123 expressing leukemic targets. When analyzing the safety profile of the CAMs against low-CD123+ endothelial target cells (≈1600 molecule/cell), we observed a lower functional activity of the CAMs as compared to the leukemic cell lines expressing the same level of CD123 molecules on their surface, with the low-affinity CAM-2 showing a major sparing capability in terms of killing activity (being the only one not statistically different from NO DNA). In conclusion, exploiting our model of affinity mutants we were able to in vitro characterize the role of the CAR density balanced with the affinity of the anti-CD123.CAR towards AML cells expressing different CD123 levels and CD123-low expressing normal tissues. In particular, the results obtained with CAM-2 suggested a potential threshold of affinity below which, even if the safety profile is preserved, the anti-leukemic efficacy would be impaired. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 8
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 4171-4171
    Abstract: Chronic Lymphocytic Leukemia (CLL) is a chronic lymphoid malignancy characterized by immune suppression that is responsible for an increase in infection susceptibility but also concurs to a reduced ability of the immune system to promote an effective response against the leukemic cells. Tumor-immunosuppressive mechanisms are essentially due to the capacity of CLL cells of modifying the surrounding microenvironment including immune effectors likely contributing to disease progression but also to limited effectiveness of current immunotherapy approaches. Lenalidomide is an immunomodulatory agent (IMID) able to induce significant long-lasting responses in CLL patients. The exact mechanism of anti-tumor activity of lenalidomide remains undefined, but it also implies the modulation of tumor microenvironment through down-regulation of critical cytokines and activation of immune effector cells. In addition, lenalidomide was shown to reverse, in vitro, defects in immunological synapse formation between T cells and CLL cells, by interfering with several cytoskeletal molecules. Chimeric antigen receptors (CARs) molecules are emerging as a powerful tool to redirect T-cell specificity against leukemia. CARs are artificial molecules constituted by an extracellular-antigen-binding domain consisting of the variable chains of a monoclonal antibody, linked together as a single chain Fv (scFV), and an intracellular signaling region, usually the zeta chain of the TCR/CD3 complex, that is immediately triggered after antigen recognition. Therefore, CARs take advantage of both the antigen binding non MHC-restricted-properties of monoclonal antibodies and of the typical T-cell mediated effector functions. Given the characteristic T cell defects occurring in vivo in CLL patients, it becomes very intriguing to explore the possibility of a novel CLL therapy combining a CAR-based immunotherapy with low doses of lenalidomide, in order to maximize the effect of the immune attack by reverting in vivo the acquired T cell defects. We studied the in vivo cytotoxic effects on the tumor microenvironment upon lenalidomide treatment utilizing the Rag2-/-γc-/--xenograft model of human CLL based on transplantation of the CLL cell line MEC1 into Rag2-/-γc-/--mice. Utilizing the CAR.CD23 tool as previously published by our group, we also performed experiments where MEC-1-trasplanted-Rag2-/-γc-/- mice were injected with CAR.CD23 T cells from CLL patients together with lenalidomide at low concentrations, uneffective in monotherapy. In these animals, a decrease of the percentage of CD19+leukemic cells was observed in all lymphoid and non-lymphoid tissues after 20 days of treatment, as compared to controls treated with CAR.CD23 T cells or lenalidomide alone. This combination resulted also in improved survival of the treated cohort (NT+lenalidomide vs CAR+lenalidomide: p 〈 0.03, n=7). The effect of the combination with low dose lenalidomide was more effective also when compared to the addition of human recombinant IL-2 as in traditional immunotherapeutic settings. In accordance to the in vivo efficacy, CAR T cells were observed in all leukemic sites suggesting an ability to migrate and home in vivo. In addition, when purified from the bone marrow CD23.CAR+T cells were still able to mount a tumor-specific cytotoxic response in vitro, reaching more than 50% of tumor lysis in both the conditions with lenalidomide and IL-2, compared to 20% of tumor lysis exerted by unmanipulated T cells. Indeed, ex vivo T cells were for the majority effector memory cells and the CD23.CAR was still expressed on their surface. These results conceivably support the use in the CLL therapeutical setting of low doses lenalidomide to improve CARs cytotoxic response and avoid the potential impairment of an effective immune response. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 9
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 3160-3160
    Abstract: Introduction Cytokine Induced Killer (CIK) cells are memory T lymphocytes which have acquired CD56 expression and Natural Killer (NK) like unrestricted cytotoxicity, following in vitro activation by anti CD3 OKT3 and IFNg and subsequent expansion with IL-2. CIK cells have demonstrated in vitro and in vivo anti tumor activity, direct intratumor homing following iv. administration and, more importantly, a very reduced Graft Versus Host (GVH) activity, in several experimental allogeneic models. Indeed we and others have demonstrated very limited GVH activity in preliminary phase I studies, with donor derived (matched) CIK cells in patients with different hematological neoplasms, previously treated by allogeneic Hematopoietic Stem Cells (HSC) transplantations and subsequently relapsed of diseases 1. Methods To better delineate the toxicity profile, as well as the potential anti tumor efficacy, of donor derived CIK cells, we prospectively studied 48 patients relapsed after allogeneic stem cell transplantation performed using either a matched related (N=28) or unrelated donor (n= 20, including 1 haplo). This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, approved by the Agenzia Italiana del Farmaco and (AIFA). The trial was registered as (EUDRACT n 2008-003185-26, ClinicalTrial.gov: NCT01186809) Results In this interim analysis, forty-eight patients (including childrens and adults) have been so far enrolled into this study protocol. The median age was 48 (range 6-67) and a diagnosis of ALL (n=9, 20%), AML (n=29, 60%), MM (n=5, 8%), HD (n=3, 6%), MPN (n=2, 4%), NHL (n=1, 2%). Reasons for being enrolled into study was a hematologic relapse in 36 (75%) or a molecular relapse in 12 (25%). The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with growing numbers of CIK cells, in groups of three patients per dose level. Since DLT was never observed (acute GVHD of grade III or more) the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to subsequent consecutive 36 patients. 4 patients died for disease progression and 1 patient developed aGVHD (grade I, skin only) during the DLI treatment and could not proceed to the planned subsequent CIK administration. Of the 43 patients who eventually received at least one infusion of CIK cells, 15 patients did not complete the program, 9 for disease progression and death, 3 for insurgence of grade II aGVHD (skin only in 2 cases, skin and gut in 1 case), 1 for hemolytic anemia, 1 for insufficient cell supply and 1 for medical decision. Overall, 28 patients received the complete cell therapy planned (58%). Overall, of the 48 patients enrolled, 5 (10%) suffered from aGVHD (1 grade I, 3 grade II, 1 grade III). During follow up, chronic GVHD was observed in 7 patients (14 %) (3 mild and 4 moderate). As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 13 (27%) patients (4 during the DLI), before the clinical response could be evaluated. A CR was observed in 9 (19 %) and a PR in 7 (14%) for an overall response rate of 16 (33%). No response was observed in 19 (39%). At 2 and 4 years, the event free survival of the 48 patients is 22% and 18%, while the overall survival is of 37% and 34%, respectively. For the small group of patients who achieved a complete response, the disease free survival is of 64% at 2 years and 51% at 4 years. By univariate analysis, survival was significantly associated to the type of relapse (molecular) (p 0.0081) since at 2 and 4 years it was and 24% and 27% vs. 71% and 71 % for patients enrolled for a hematologic or a molecular relapses, respectively. By multivariate analysis, the type of relapse remained the only significant predictor of survival (0.0160 p value). Conclusion This study shows the feasibility of CIK preparation and administration as well as the relatively low toxicity of the program (10% aGVHD grades I-III) in spite of the fact that 20 patients received cells from matched unrelated donors. Finally, the study offers the suggestion that CIK cells may be efficacious to treat post-transplant relapse. 1 Introna M. et al, Haematologica, 2007, 92, 7, 948. Figure 1. Figure 1. Disclosures Introna: roche: Research Funding. Rambaldi:Roche: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Celgene: Research Funding; Pierre Fabre: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 10
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 3538-3538
    Abstract: B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of mature B-lymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect T-cell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and anti-CD19/anti-CD20 CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral responses. Selective CD23 expression on B-CLL cells renders it an optimal target to design a specific CAR. A new CD23-targeting CAR to redirect T cells against CD23+ B-CLL has been generated. After transduction, modified T cells were tested for cytotoxicity against different CD23+-targets, using a classic chromium release assay and for specific cytokine release by multiplex flow cytomix assay. The anti-CD23 CAR was stably expressed by healthy donor-derived primary T cells after transduction (average expression,20%;range,10%–60%;n=10) and conferred them a strong cytotoxicity against CD23+ tumor cell lines: Epstein Barr Virus transformed lymphoblastoid cell line (EBV-LCL) (average lysis, 50%; range 15%–70%, at 40:1 Effector:Target (E:T) ratio; n=5); Bjab and Jeko cell lines transduced with human CD23 antigen (average lysis, 60%; range, 20%–75%, at 40:1 E:T ratio; n=3). On the contrary, anti-CD23 transduced T-cells displayed no relevant killing versus normal B cells (average lysis, 8%; range, 1%–15% at 40:1 E:T ratio; n=3), differently from anti-CD19 CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. T cells from B-CLL patients were also efficiently transduced with the anti-CD23 CAR (average expression, 80%; range, 70%–90%; n=3) and redirected specifically toward autologous blasts (average lysis, 29%; range, 21%–35% at 20:1 E:T ratio; n=3), without being inhibited by soluble CD23-enriched autologous plasma. Moreover, we demonstrated that expression of the anti-CD23 CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after 48h stimulation with irradiated EBV-LCL at 1:1 ratio, both in healthy donors (n=3) and B-CLL patients (n=2). Anti-CD23 CAR expressing T cells from healthy donors secreted 5.5-fold more INF-gamma (3079 pg/ml vs 561pg/mL, p=0.05) and 11-fold more TNF-alpha (187.17 pg/ml vs 16.53 pg/mL, p=0.05), 147-fold more IL-5 (147 pg/ml vs 0 pg/mL, p=0.05) and 13-fold more IL-8 (590 pg/ml vs 43.24pg/mL, p=0.05), compared to non transduced T cells (n=3). In line with these findings, T cells expressing anti-CD23 CAR from B-CLL donors secreted 8.8-fold more INF-gamma (2988 pg/ml vs 337pg/mL, p=0.05) and 17-fold more TNF-gamma (187.17 pg/ml vs 17.34 pg/mL, p=0.05); 25.8-fold more IL-5 (3483.14 pg/ml vs 134.785 pg/mL, p=0.05), 173-fold more IL-8 (2154 pg/ml vs 12.415 pg/mL, p=0.05), compared to non transduced T cells. Altogether these results suggest that for the potentiality to get selective and potent killing of tumor cells, while sparing normal B cells, and for the capability to induce the selective release of immunostimulatory cytokines, CD23-targeting through a specific CAR holds great promises for adoptive immunotherapy of B-CLL.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
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