Publications by authors named "Elisa Gotti"

10 Publications

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Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19 tumors.

Cytotherapy 2018 08 7;20(8):1077-1088. Epub 2018 Aug 7.

Center of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy. Electronic address:

Background: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19 malignancies.

Methods: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs.

Results: CB-CIKs, like PB-CIKs, were mostly CD3 T cells with mean 45% CD3CD56 and expressing mostly TCR(T cell receptor)αβ with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4 cells, mostly CD56, as well as a greater proportion of naïve CCR7CD45RA and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8 cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19 tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph CD19 acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 10 CIK/kg.

Discussion: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.
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http://dx.doi.org/10.1016/j.jcyt.2018.06.003DOI Listing
August 2018

Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance.

Cytotherapy 2018 02 12;20(2):262-270. Epub 2017 Dec 12.

Centre of Cellular Therapy "G. Lanzani", Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy.

Background: We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs).

Methods: For sterility tests 14-day cultures were performed in culture media detecting aerobic and anaerobic microorganisms.

Results: In this study, 22/1643 (1.3%) of apheretic products for autologous or allogeneic HSCT were contaminated, whereas 14/73 bone marrow (BM) harvests (17.8%) were positive. In 22 cases, the contaminated HSCs were infused to patients, but there was no evidence of any adverse impact of contamination on the hematologic engraftment or on infections. Indeed none of the five positive hemocultures detected in patients following infusion could be linked to the contaminated stem cell product. Our Cell Factory also generated 286 ATMPs in good manufacturing practice (GMP) conditions since 2007 and all final products were sterile. In three cases of mesenchymal stromal cell expansions, the starting BM harvests were contaminated, but the cell products at the end of expansion were sterile, presumably thanks to the presence of an antibiotic in the culture medium.

Discussion: The decreased rate of contamination of cell harvests observed with time suggests that routine sterility testing and communication of the results to the collecting centers may improve clinical practices. Furthermore, we recommend the use of antibiotics in the medium for ATMP expansion, to decrease the likelihood of expanding microorganisms within clean rooms. Finally we discuss the costs of sterility testing of ATMPs by GMP-approved external laboratories.
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http://dx.doi.org/10.1016/j.jcyt.2017.11.009DOI Listing
February 2018

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Biol Blood Marrow Transplant 2017 Dec 13;23(12):2070-2078. Epub 2017 Jul 13.

USC Hematology and Bone Marrow Transplant Unit ASST Papa Giovanni XXIII Bergamo, Bergamo, Italy; Università degli Studi di Milano, Milan Italy.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.
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http://dx.doi.org/10.1016/j.bbmt.2017.07.005DOI Listing
December 2017

Kinetics of the use of cryopreserved autologous stem cell grafts: a GITMO-SIDEM survey.

Cytotherapy 2014 Jan 26;16(1):101-10. Epub 2013 Oct 26.

Hematology Clinic, Marche Polytechnic University, Ancona, Italy. Electronic address:

Background Aims: Hematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient.

Methods: We conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU.

Results: In our cohort, 79% of patients collected >5 × 10⁶ CD34+ cells/kg, and 3.4% collected <2 × 10⁶ CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 × 10⁶ CD34/kg and lower age groups.

Conclusions: These data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.
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http://dx.doi.org/10.1016/j.jcyt.2013.07.001DOI Listing
January 2014

Ofatumumab is more efficient than rituximab in lysing B chronic lymphocytic leukemia cells in whole blood and in combination with chemotherapy.

J Immunol 2013 Jan 5;190(1):231-9. Epub 2012 Dec 5.

Laboratory of Cellular Therapy G. Lanzani, Division of Hematology, Ospedali Riuniti di Bergamo, 24128 Bergamo, Italy.

Ofatumumab (OFA) is a human anti-CD20 Ab approved for treatment of fludarabine-refractory B chronic lymphocytic leukemia (B-CLL). The efficacy of different immunotherapeutic strategies is best investigated in conditions that are as physiologic as possible. We have therefore compared the activity OFA and rituximab (RTX), alone or in combination with chemotherapeutic agents in unmanipulated whole blood assays, using flow cytometry. OFA (10-100 μg/ml) lysed B-CLL targets in whole blood more efficiently and with faster kinetics than RTX, with a mean 56% lysis at 24 h compared with 16%. This activity of OFA was fully complement dependent, as shown by >99% inhibition by anti-C5 Ab eculizumab and a lack of NK cell activation in whole blood. OFA-mediated NK cell activation was blocked by complement. OFA-mediated lysis could be increased an additional 15% by blocking CD55 and CD59 complement inhibitors. Interestingly, OFA-mediated lysis correlated significantly with CD20 expression levels (r(2) = 0.79). OFA showed overlapping dose response curves similar to those for RTX in phagocytosis assays using either human macrophages or neutrophils. However, phagocytosis was inhibited in the presence of serum or whole blood. Finally, combined treatment with mafosfamide and fludarabine showed that these therapeutic drugs are synergistic in B-CLL whole blood assays and show superior activity when combined with OFA compared with RTX. These results confirm in B-CLL samples and in physiologic conditions the superior complement mediated cytotoxicity induced by OFA alone compared with RTX, the lack of NK cell activation, and phagocytosis in these conditions and suggest effective chemoimmunotherapy strategies using this new generation anti-CD20 Ab.
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http://dx.doi.org/10.4049/jimmunol.1202645DOI Listing
January 2013

Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells.

Stem Cells Dev 2013 Mar 21;22(5):772-80. Epub 2012 Dec 21.

Mario Negri Institute for Pharmacological Research, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Bergamo, Italy.

Bone marrow-mesenchymal stem cells (BM-MSC) ameliorate renal dysfunction and repair tubular damage of acute kidney injury by locally releasing growth factors, including the insulin-like growth factor-1 (IGF-1). The restricted homing of BM-MSC at the site of injury led us to investigate a possible gene-based communication mechanism between BM-MSC and tubular cells. Human BM-MSC (hBM-MSC) released microparticles and exosomes (Exo) enriched in mRNAs. A selected pattern of transcripts was detected in Exo versus parental cells. Exo expressed the IGF-1 receptor (IGF-1R), but not IGF-1 mRNA, while hBM-MSC contained both mRNAs. R- cells lacking IGF-1R exposed to hBM-MSC-derived Exo acquired the human IGF-1R transcript that was translated in the corresponding protein. Transfer of IGF-1R mRNA from Exo to cisplatin-damaged proximal tubular cells (proximal tubular epithelial cell [PTEC]) increased PTEC proliferation. Coincubation of damaged PTEC with Exo and soluble IGF-1 further enhanced cell proliferation. These findings suggest that horizontal transfer of the mRNA for IGF-1R to tubular cells through Exo potentiates tubular cell sensitivity to locally produced IGF-1 providing a new mechanism underlying the powerful renoprotection of few BM-MSC observed in vivo.
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http://dx.doi.org/10.1089/scd.2012.0266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578372PMC
March 2013

Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate.

Cytotherapy 2011 Aug 18;13(7):786-801. Epub 2011 Mar 18.

Laboratory of Cell Therapy G. Lanzani, USC Hematology, Ospedali Riuniti di Bergamo, Bergamo, Italy.

Background Aims: Mesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC).

Methods: Sections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3).

Results: We isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10(10) cells (ranging from 1.0 × 10(10) to 29.0 × 10(10)). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo.

Conclusions: UC constitute a convenient and very rich source of MSC for the production of third-party 'clinical doses' of cells under good manufacturing practice (GMP) conditions.
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http://dx.doi.org/10.3109/14653249.2011.563294DOI Listing
August 2011

Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab.

J Immunol 2011 Mar 4;186(6):3762-9. Epub 2011 Feb 4.

Laboratory of Cellular Therapy G. Lanzani, Division of Hematology, Ospedali Riuniti, 24128 Bergamo, Italy.

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.
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http://dx.doi.org/10.4049/jimmunol.1000303DOI Listing
March 2011

The washouts of discarded bone marrow collection bags and filters are a very abundant source of hMSCs.

Cytotherapy 2009 ;11(4):403-13

Laboratory of Cell Therapy G. Lanzani, USC Hematology, Ospedali Riuniti di Bergamo, Bergamo, Italy.

Background Aims: Human multipotent mesenchymal stromal cells (hMSC) are considered good candidates for a growing spectrum of cell therapies. We have validated a protocol that makes use of the washouts of discarded collection sets, left over at the end of the filtration of bone marrow (BM) explants performed for hematopoietic stem cell (HSC) transplantation.

Methods: The method consists of direct plating of cells without density-gradient isolation followed by two detachment steps and expansion in 5% human platelet lysate (hPL).

Results: In a median of 26 days, 14 bags for adult patients and nine bags for pediatric patients for a standard dose of 1x10(6) hMSC/kg body weight could be prepared from the expansion of a fraction of the cells recovered from seven independent washouts. Moreover, 151 vials could be frozen from the remaining cells. The theoretical full expansion of all the frozen vials (validated by the expansion of two independent vials) could have allowed the production of 173 bags for adults and 348 bags for pediatric patients.

Conclusions: The washouts of discarded bags and filters left over at the end of routine BM explants filtration are a very abundant source of hMSC precursors that can be easily utilized for clinical applications.
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http://dx.doi.org/10.1080/14653240902960437DOI Listing
September 2009

M2 macrophages phagocytose rituximab-opsonized leukemic targets more efficiently than m1 cells in vitro.

J Immunol 2009 Apr;182(7):4415-22

Ospedali Riuniti di Bergamo, Italy.

Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 microg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcgammaRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-gamma/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-gamma had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.
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http://dx.doi.org/10.4049/jimmunol.0713732DOI Listing
April 2009