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  • American Society of Hematology  (10)
  • 2010-2014  (10)
  • 1
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    Stable Expression Of Chimeric Antigen Receptors (CARs) By Sleeping Beauty-Mediated Gene Transfer and Efficient Expansion Of Leukemia-Specific Cytokine-Induced Killer (CIK) Cells
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 1663-1663
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1663-1663
    Abstract: Gene-manipulation of effector T cells with CARs has recently turned into a powerful tool to redirect antigen specificity for adoptive immunotherapy of tumors. Although promising clinical efficacy has been demonstrated, critical issues concerning the profile of efficacy, safety and feasibility of cell manufacturing and gene therapy still remain partially unsolved. In order to rescue the concerns associated to viral vectors that limit so far their clinical applicability, we have explored here the use of the latest generation Sleeping Beauty Transposon-mediated gene transfer. Since current protocol of nucleofection associated with Transposons impaired subsequent expansion and vitality of modified cells, we generated and propagated CAR+ cytokine-induced killer (CIK) cells with the purpose of optimizing cell expansion. Actually, our experience with CIK cells clearly proved that the production of large numbers of unmanipulated allogeneic cytotoxic effector T cells is feasible under clinical-grade conditions, and repeated infusions in patients are safe and well tolerated (Introna et al., Haematologica 2007). Using an optimized stimulation protocol based on the addition of accessory cells, irradiated PBMCs, after nucleofection, we genetically modified CIK cells to express two distinct 3rd generation CARs (CD28/OX40/TCR zeta) specific for acute myelogenous leukemia (AML) CD123+ or acute lymphoblastic leukemia (ALL) CD19+ blasts. With this system, the average transfection at 24hours was 54.6% (±8.6, n=8) and mean survival percentage was 63.8% (±8.8, n=12). Nucleofection did not affect the phenotype of CIK cells, and, most importantly, the addition of accessory cells was effective in inducing T-cell expansion, with a fold increase of 39.4±9.8 within 3 weeks, sufficient to be translated into adoptive cell therapy clinical protocols. Transposed CIK cells displayed stable expression of CD123-CAR or CD19-CAR with a frequency of modified cells of 48.9%±3.3 (n=11) and 47%±6.4 (n=4), respectively. Efficient lysis of leukemic cell lines and primary blasts was observed and cytotoxic degranulation was associated to CAR expression, indicating a specific target recognition by the CAR. Interestingly, CAR triggering by the encounter with the specific antigen expressed by leukemic cells promoted specific cytokine secretion and proliferation, suggesting activation and selection of modified CIK cells upon encounter with cancer cells. Finally, preliminary insertion-site analysis by LAM-PCR confirmed the polyclonal profile of integrations in the genome of Sleeping Beauty system. These Results provide pre-clinical evidences of efficient transfection of CD123- and CD19- CARs using Sleeping Beauty-mediated gene transfer, specificity of action and improvements in Methods of expansion of cytotoxic effector T cells. The development of an adoptive cell therapy protocol based on a reproducible clinical-grade method of expansion and an innovative gene transfer process will be fundamental to envisage clinical protocols to control relapse in leukemic patients and to improve the range of applications of such novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.1663.1663
    RVK:
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    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 2
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    Understanding the Immunomodulatory Effect of Mesenchymal Stem Cell Infused In Transplanted Patients with Steroid-Refractory GvHD
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 2306-2306
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2306-2306
    Abstract: Abstract 2306 In the last few years the usage of third party mesenchymal stem cells (MSC) as therapy for steroid-refractory Graft versus Host Disease (GvHD) is constantly increasing and holds big promises. Nevertheless, at our knowledge, studies on MSC efficacy have been scarcely corroborated by biological analysis of patient response to cell infusion. Here, we report the immunological monitoring of 8 patients (7 male, 1 female; aged 4 to 33 years), with steroid-refractory GvHD (grade II to III), who received MSCs, between August 2009 and June 2010. GvHD presented as acute in 6 cases and chronic in 2 cases. In 5 cases GvHD occurred as a single organ pathology (2 skin, 2 gut, 1 liver), while in 3 cases GvHD had multi-organ involvement (1 liver and oral mucosa, 1 skin and oral/ocular mucosa, 1 skin, gut and liver). All patients received 2 to 3 MSC infusions from third party donors aiming at 1 × 106/kg recipient body weight MSCs for each infusion. After MSC therapy, 2 patients showed complete response, 3 patients showed partial response, whereas 3 patients did not respond to MSC infusion. To better comprehend the immunomodulatory effects of MSC infusions, we studied GvHD plasmatic markers, inflammatory cytokines and CD4+ T-cell subsets circulating in the peripheral blood (PB) of enrolled patients before MSC infusion and at day 7, 14 and 28 after cell therapy. In accordance with clinical observations, in patients responding to MSC infusions, we observed a dramatic decrease of three validated GvHD plasmatic markers TNFRI, IL2Rα and elafin (Paczesny S et al. Blood 2009) to the mean levels of Healthy Donors (HD). In particular, at day 28 after therapy, TNFRI and IL2Rα levels decreased of 2 times (range=1.9-2.4 and range=1.4-2.8, respectively) and elafin levels decreased of 2.5 times (range=1.7-3.6). Partially responding patients showed a transient decrease of TNFRI, IL2Rα and elafin levels, while non responding patients showed stable or even increasing levels of all analysed markers. Moreover, we investigated the effect of MSC infusion on lymphocyte counts. We demonstrated that patients responding to MSC infusion, oppositely to non responders, strongly decreased total and CD4+ lymphocyte counts in the PB (mean total T-cell Fold Decrease (FD)=11.85, range=1.3-116; mean CD4+ T-cell FD=12, range=1.5-116). Interestingly, after MSC infusion, CD4+ T-cell subsets changed significantly: Tregs increased and Th1 and Th17 populations decreased, and a new CD4+ cell subset balance was observed starting from day 7 after therapy. In particular, the mean FD of Th1/Treg ratio was 4.1 (range=4-4.2) and the mean FD of Th17/Treg ratio was 4.7 (range=3.3-6). Correspondingly, patient symptoms also gradually improved, suggesting an association between GvHD clinical course and CD4+ T-cell imbalance, reverted by MSCs in responding patients. In partially responding patients Th1/Treg and Th17/Treg showed a transient decreased and even slightly increased in the case of non responding patients. In accordance with the decrease of Th1 CD4+ T cells in the PB of patients responding to MSC infusion, we observed a valuable decrease of IFNγ plasma concentrations (mean FD=48, range=30-65 in complete responders), which reached the levels typical of HD. In summary, despite its limited size, the present study suggests that MSCs, upon infusion, are able to convert an inflammatory environment to a more physiological one, both at a cellular level, promoting the expansion of circulating Tregs, and at a molecular level, diminishing inflammatory cytokines. Further studies on a larger group of patients, clarifying the mechanisms of action used in vivo by MSC to tune ongoing allo-reactions, will be fundamental to provide the rationale for improving current clinical trials. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.2306.2306
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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  • 3
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    A New Chimeric Antigen Receptor (CAR) Targeting the CD23 Antigen Expressed by Chronic Lymphocytic Leukemia (B-CLL) Cells
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 2446-2446
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2446-2446
    Abstract: Abstract 2446 B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of 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 immunity. Selective CD23 expression on B-CLL cells renders this molecule an optimal target to design a specific CAR. We have generated a novel CD23-targeting CAR to redirect T cells against CD23+B-CLL. Transduced T cells were tested for cytotoxicity against different CD23+-targets, using a classic 51Chromium release assay, and for specific cytokine release, by multiplex flow cytomix assay. T cells from B-CLL patients were efficiently transduced with the anti-CD23.CAR (average expression 68%, n=10) and redirected specifically toward autologous blasts (average lysis 58%, n=5). On the contrary, anti-CD23 transduced T-cells did not displayed any relevant killing versus normal B cells (average lysis 13%, n=3), differently from anti-CD19.CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. Moreover, anti-CD23.CAR redirected T lymphocytes derived from both healthy donors (HD) and B-CLL patients displayed a specific lytic activity against CD23+EBV-LCLs, even in presence of soluble CD23 enriched plasma without being inhibited (average lysis with no plasma 67%; average lysis with 25% of CD23 enriched plasma 79%; average lysis with 50% of CD23 enriched plasma 88%, n=3). We also demonstrated that the expression of the anti-CD23.CAR caused a significant increase in cytokine release from transduced in vitro activated T cells after a 48h stimulation with CD23+ targets. B-CLL derived CD23.CAR-expressing T cells (n=3) secreted 4-fold more INF-gamma, and 1445-fold more TNF-beta, compared to non transduced T cells. Interleukin-2 was also released (average release 2681 pg/mL, n=3) and sustained the antigen-dependent proliferation of CD23.CAR+T cells. To confirm in vivo the in vitro data, we tested NT and CD23.CAR transduced T cells in a recently published xenograft model of B-CLL (Ref biblio, primo nome, giornale, anno). This model is based on the intravenous or subcutaneous injection of the established human B-CLL cell line MEC1 into Rag2−/− gammac−/− mice, which lack not only B and T cells, but also natural killer (NK) cells, thus presenting a profound immunosuppressive environment leading to an high efficiency of both B-CLL and T-cell engraftment. Moreover, this model reproduces the systemic involvement of the disease and it closely resembles the aggressive form human B-CLL, representing an optimal experimental setting to test the efficacy of new therapeutic agents. In the first set of our experiments, 8 weeks-old male mice were subcutaneously injected with 10*106 MEC1 cells in the left flank. Then, mice bearing an established tumor were treated intravenously with a single dose of NT or engineered CD23.CAR T cells (2*106), without any addition of exogenous IL-2. Animals were monitored twice a week for weight and tumor growth (measuring three perpendicular diameters), and sacrificed when the mean tumor volume reached a dimension of 31000 mm3, before presenting clinical signs and symptoms. Compared with NT-treated mice, the infusion of CD23.CAR+T cells resulted in a significant delay in tumor growth, as measured by tumor volume diameter/day (CD23.CAR+ T cells vs NT T cells: p=0.04 at day 12; n=3). In conclusion, our results suggest that CD23.CAR-redirected T cells provide cytotoxic activity against CD23+ B-CLL cells in vitro and in vivo, while sparing normal B lymphocytes, as compared to other available CARs targeting pan-B-cell antigens, such as CD19 and CD20. These results are encouraging and demonstrate the feasibility of generating CD23.CAR+ T lymphocytes for adoptive T-cell therapy of patients with B-CLL. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.2446.2446
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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  • 4
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    Chimeric Antigen Receptor for Specific Targeting of Acute Myeloid Leukemia
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 4225-4225
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 4225-4225
    Abstract: Abstract 4225 Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after conventional chemotherapy regimens. Therefore it is necessary to develop novel alternative approaches. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial T-cell receptors constituted by a specific antigen-binding domain, and a signaling region, that, upon antigen recognition, leads to T-cell activation, and lysis of the target cells. AML is a potential optimal target for CAR strategy because of the over-expression of a number of surface antigens like CD33, CD123. Since CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently emerged as new potential attractive molecules based on its differential expression pattern, being still wildly overexpressed by AML population, and at the same time less expressed on HSPCs. Here we describe the in vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK) cells genetically modified to express CAR molecules specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted NSG mice (median of hCD45+CD33+ 0.6% before treatment), genetically modify T cells had a potent antitumor effect. Indeed, the bone marrow of control untreated animals or mice treated with un-manipulated CIK cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-ζ and 8/10 anti-CD123-CD28-OX40-ζ treated mice we couldn't detect any AML cells. Similar results have been obtained when the treatment via T cell injection start when high AML burden has been obtained (median of hCD45+CD33+ 70% before treatment). One week after the last CIK's injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK cells and with anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells respectively. We performed secondary transplantation on the residual AML cells present in these animals and mice were treated again with transduced CIK cells. Residual AML cells were still sensitive to CARs approach, leading once again to an almost complete eradication of the disease (median level of hCD45+CD33+ engraftment was 98%, 0.02% and 0.04% respectively for untreated mice, anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells). Furthermore, a fundamental issue was to determine the safety profile of such approach against normal hematopoietic precursors. In untreated mice injected with primary cord blood derived CD34+ cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with un-manipulated, anti-CD33-CD28-OX40-ζ or anti-CD123-CD28-OX40-ζ transduced CIK-cells the levels of human compartment was 40%, 11.7% and 26.3% respectively. Moreover when we consider specifically the CD34+CD38- compartment, enriched in HSC, the level of engraftment was 1.92%, 1.02%, 0.55% and 0.83%. Secondary transplantations are now ongoing to give a more complete profile about the remaining HSC repopulating capability after treatment. To more closely mimic a physiological context, similar experiments are ongoing using mice engrafted with normal adult bone marrow instead of umbilical cord blood. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy particularly in the context of minimal residual disease in high-risk transplanted AML patients. Moreover CAR approach could be potentially used to treat patients resistant to conventional chemotherapeutic approaches or for whom high dose chemotherapy treatment could not be proposed. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.4225.4225
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 5
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    Vaccination - a Novel Strategy to Improve the Persistence of CD19CAR Transduced T-Cells in Relapsed Paediatric ALL: Preliminary Results from the CD19TPALL Study
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 383-383
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 383-383
    Abstract: Introduction Patients with acute lymphoblastic leukaemia (ALL) relapsing after allogeneic stem cell transplantation (SCT) have a dismal prognosis. Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) incorporating co-stimulatory domains for improved persistence and expansion report unprecedented anti-leukemic responses. However, responses are associated with Cytokine Release Syndrome (CRS) due to supra-physiological activation of the redirected T-cells. As an alternative, we studied use of donor-derived Epstein Barr virus (EBV)-specific T cells (CTL) transduced with a 1st generation CD19CAR as effectors, relying on signalling through the endogenous T cell receptor (TCR) to drive more physiological proliferation and persistence. This has enabled us to investigate a novel strategy to facilitate the expansion/persistence of CD19CAR T cells by vaccination with irradiated donor-derived, EBV transformed lymphoblastoid cell lines (LCL). We are conducting a European multi-centre phase I/II study of this approach in patients with pediatric ALL relapsing after SCT and report our interim findings. Methods Donor-derived EBV-specific CTL were generated from 80mls donor blood by repetitive stimulation with LCL, followed by transduction with an SFG retroviral vector encoding a CD19CAR consisting of the FMC63 single chain Fv linked to a CD3ζ endodomain. Patients were eligible for CD19CAR CTL therapy either pre-emptively if they became MRD-positive ( 〉 5 x 10-4 in BM) within the 1st year post-SCT or prophylactically at day 60-70 post-2nd SCT. All patients had early withdrawal of immunosuppression and received lymphodepletion with fludarabine 90 mg/m2. Patients with detectable residual disease also received cytoreduction with vincristine/dexamethasone prior to infusion of cryopreserved CD19CAR CTL. Persistence of CAR CTL was measured by quantitative PCR and flow cytometry of blood. Disease status was assessed by morphology and IgH MRD analysis on bone marrow samples. The study design incorporated an interim analysis, allowing for vaccination with irradiated LCL if CD19CAR CTL were not detectable in 50% of patients at 2 months post-infusion. Results So far, 20 patients have been recruited (14 pre-emptive, 6 prophylactic arm) and 7 patients treated (3 pre-emptive, 4 prophylactic). The infused cell dose was 2 x 108/m2 in 6 patients and 4 x 107/m2 in the other. CD19CAR expression varied from 12.1-58.9%. No grade 3-5 toxicity was noted. In particular, no CRS, neurotoxicity or graft versus host disease (GVHD) attributable to CD19CAR CTL was seen. B-cell depletion was transient, lasting 1-2 months. In terms of disease response, 2 patients treated prophylactically remain in MRD negative remission after 3 and 17 months’ follow-up. A further patient showed transient clearance of BM MRD following immunotherapy in association with EBV viremia. He subsequently relapsed but has stable disease after retreatment with CD19CAR CTL with LCL vaccination. The other 4 patients had disease progression between 2 weeks and 3 months post-CD19CAR CTL infusion. At a median follow-up of 8 months, 2 patients have died of relapse, 3 are alive with disease and 2 remain disease-free. A planned interim analysis of the initial 6 patients treated with CD19CAR CTL alone showed poor expansion/persistence of CD19CAR CTL which were only detectable in the blood in 1 patient up to 28 days post-infusion. This may reflect that only 1 patient had EBV viremia at the time CD19CAR CTL were infused. In view of this, a second trial cohort received subcutaneous vaccination with irradiated, donor-derived LCL at 2 days before and at 1 and 2 months following CD19CAR CTL infusion to provide signalling through the endogenous EBV-specific TCR. So far, 2 patients have been vaccinated and a 3rd is planned shortly. Data on the effect of vaccination on CD19CAR CTL expansion/persistence will be presented. Conclusions This ongoing study shows safety of adoptive immunotherapy with donor EBV CTL transduced with a 1st generation CD19CAR in paediatric patients with ALL relapsing post allo-SCT. However, in the absence of a co-stimulatory domain in vivo expansion and persistence of transferred CTL is poor. We are investigating whether vaccination with irradiated, donor-derived EBV LCL improves persistence and efficacy of CAR transduced T cells and initial data on this approach will be presented Disclosures Pule: Cellectis: Martin Pule's laboratory receives funding for contract research from Cellectis Therapeutics Other.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.383.383
    RVK:
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    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 6
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    Safe and Effective Treatment of Graft Versus Host Disease with Platelet Lysate-Expanded Human Mesenchymal Stromal Cells: A Phase 1 Study On 47 Adult and Pediatric Patients
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 743-743
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 743-743
    Abstract: Abstract 743 Background: Acute Graft versus host disease (aGvHD) is a severe complication of allogeneic hematopoietic stem cell transplantation (HSCT). Conventional treatment with high dose steroids fails to achieve a complete and sustained response in more than 50% of patients. Several second line treatments have been described but none of these can be considered superior or a standard of care (Paul J. Martin et al, BBMT 2012). Among these treatments, the use of third party mesenchymal stromal cells (MSC) has been proposed (LeBlanc et al, Lancet 2008). In this study, we assessed the safety and efficacy of third party human MSC, in a prospective, multicenter, phase I study (EudraCT 2008–007869-23). Methods: Forty-seven patients with steroid-resistant, acute or chronic grade II-IV GvHD were enrolled into this study. Human MSC were obtained from bone marrow harvests of healthy donors and expanded in vitro using serum free medium supplemented with human platelet lysate (Capelli C et al, BMT, 2007; Capelli C. et al, Cytotherapy 2009). In vitro expanded MSC were produced in two officially authorized Cell Factories and tested in four Italian Hematology Units. The primary endpoint of this study was the safety. Secondary endpoints were the response of GvHD (evaluated 28 days after the last MSC infusion), as well as the overall survival and transplant-related deaths. Blood samples were periodically collected before and after MSC infusion to measure plasma levels of IL2Ralpha by ELISA, as previously described by our group (Dander E et al, Leukemia 2012). Results: Between August 2009, and June 2012, 47 patients (16 children, 31 adults, median age 25.5 years, range 1 to 67) were treated. The median dose of infused MSC was 1.5×106 cells per kg bodyweight. Enrolled patients presented with aGvHD in 37 cases, chronic overlap syndrome in 7 cases, and chronic classic GvHD in 3 cases. Fifteen pts had grade II GvHD, 23 grade III and 9 grade IV, according to NIH criteria. In 17 cases GvHD involved a single organ, in 24 cases 2, and in 6 cases 3 organs. Prior to MSC infusion 22 patients had received only high dose steroids, 12 patients received one cycle of pentostatin (1 mg/kg bodyweight for 3 days, Schmitt T. et al BMT, 2011: 46 580–585), while 13 received other conventional immunosuppressants. Patients received a median of 3 MSC infusions (range 1 to 8). No side effects were registered immediately after MSC infusion and no complications were lately referred as MSC-related. Overall, in 30 patients (63.8%) a clinical response of GvHD was registered. Thirteen of these patients (27.6%) had a complete response and 17 (36.1%) a partial response to treatment. Twenty-two of the 30 responding patients did not require further lines of immunosuppression after MSC infusion. Response was significantly more likely in patients exhibiting grade II GvHD versus those exhibiting more severe gradings (87.5% vs. 51.6%, p = 0.02) and in patients receiving MSC in a time interval of 30 days from the onset of GvHD (75.9% vs. 43.7%, p= 0.05). Current median follow up for this cohort is 200 days (range 30–1066). Responders show a significant lower transplant-related mortality (10.0% vs. 88.2%, p 〈 0.05) and a better overall survival probability than non responders (23.3% vs. 88.2%, p 〈 0.05, Fig. 1). Within the limit of a small subgroup analysis, adult patients receiving pentostatin before MSC had an apparent better response and survival (65% vs 27%, at 1 year), without an increased risk of infections. Measurements of plasmatic levels of IL2Ralpha, when comparing responders vs non-responders patients, showed a statistically significant difference in terms of fold decrease of the marker (p=0.027), corroborating clinical results. Similarly, a significant trend of fold decrease change (p=0.058) was observed when comparing responding patients receiving MSC within or after 30 days from the onset of the disease, in line with clinical results. Conclusions: This study confirms that human MSC prepared in academic cell therapy facilities may represent a safe and effective treatment of patients with steroid-refractory GvHD. Plasmatic inflammatory markers may help in evaluating and monitoring of clinical response. The sequential or combined administration of MSC and other immunosuppressants, such as pentostatin, is equally safe and feasible and deserves further investigation. We suggest to consider the use of MSC promptly, as early as possible, after steroid failure. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.743.743
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    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 7
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    Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 3753-3753
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 3753-3753
    Abstract: Abstract 3753 Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.3753.3753
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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  • 8
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    Targeting of Acute Myeloid Leukemia by Cytokine-Induced Killer Cells Redirected with a Novel CD123-Specific Chimeric Antigen Receptor.
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 3010-3010
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 3010-3010
    Abstract: Abstract 3010 Current therapeutic regimens for Acute Myeloid Leukemia (AML) are still associated with high rates of relapse. In the last years, great interest has been focused on the identification of surface molecules that are preferentially expressed by AML cells and leukemic stem cells (LSCs), in order to selectively target the tumor population, whilst sparing the normal counterpart of hematopoietic stem/progenitor cells (HSPC), and possibly impeding disease recurrence. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents a valid and innovative cell therapy approach for hematological malignancies. In this study we developed a new CAR molecule specific for the IL-3Rα (CD123) target antigen, which is overexpressed on AML blasts, CD34+ leukemic progenitors, and leukemic stem cells (AML-LSCs) compared to normal hematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine Induced Killer (CIK) cells, ex-vivo expanded T cells with spontaneous antitumoral activity, were transduced with an SFG-retroviral vector encoding an anti-CD123.CAR and CAR functionality has been evaluated by short-term cytotoxicity assay. Transduced CIK cells strongly killed CD123+ THP-1 cell line (60%±5.4%, Effector:Target –E:T- ratio of 5:1, n=3), as well as primary AML blasts (59%±5.4%, E:T ratio of 3:1, n=4). With the aim to better characterize the ability of anti-CD123.CAR+CIK cells to kill leukaemia cells over time we performed long-term cytotoxicity assay, observing a leukemic cell recovery for THP-1 of 3.5%±1.5% (n=5) and for primary AML cells of 2.4%±1.4% (n=3) when co-cultured with CIK cells expressing anti-CD123.CAR, compared to an average target survival of up to 80%, when co-cultured with unmanipulated (NT) CIK cells. Interestingly, secondary colonies experiments after co-culture of healthy donor cord blood-derived HSPCs (Lin-) with anti-CD123.CAR+CIK cells demonstrated that this newly generated CAR molecule better preserved the normal haematopoietic reconstitution in contrast to a previously generated anti-CD33.CAR (total number of colonies of 146.8±6.6, 66.4±5.1, 117.6±4.6, for Lin- cells co-cultured with NT CIK cells, anti-CD33.CAR+CIK cells, anti-CD123.CAR+CIK cells respectively, n=4), while keeping identical cytotoxicity profile towards AML. Furthermore, a limited killing of normal CD123 expressing monocytes and CD123-low expressing endothelial cells was measured, accompanied by a lesser release of stimulatory cytokines such as IFN-gamma, TNF-alfa and TNF-beta when compared to the levels released after stimulation with CD123+ leukemic cells (THP-1 and AML), thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CAR strongly enhances anti-leukemic CIK functions towards AML, while sparing HSPCs and normal CD123-expressing tissues, paving the way for the development of novel immunotherapy approaches for the treatment of resistant forms of AML, particularly for a precocious intervention in presence of minimal residual disease, in the context of early relapse after HSCT. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.3010.3010
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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    In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor
    American Society of Hematology ; 2011
    In:  Blood Vol. 117, No. 18 ( 2011-05-05), p. 4736-4745
    Details
    In: Blood, American Society of Hematology, Vol. 117, No. 18 ( 2011-05-05), p. 4736-4745
    Abstract: Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19+CD5+CD20dim B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR+ T cells showed specific cytotoxic activity against CD23+ tumor cell lines (average lysis 42%) and primary CD23+ CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR+ T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR+ T cells. Redirected T cells were also effective in vivo in a CLL Rag2−/−γc−/− xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR+ T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR+ T cells represent a selective immunotherapy for the elimination of CD23+ leukemic cells in patients with CLL.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2010-10-311845
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 10
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    Low-Dose Lenalidomide Improves CAR-Based Immunotherapy In CLL By Reverting T-Cell Defects In Vivo
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 4171-4171
    Details
    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
    URL: Article
    DOI: 10.1182/blood.V122.21.4171.4171
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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