Publications by authors named "Ettore Biagi"

63 Publications

Sleeping Beauty-engineered CAR T cells achieve antileukemic activity without severe toxicities.

J Clin Invest 2020 11;130(11):6021-6033

Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione MBBM, Monza, Italy.

BACKGROUNDChimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability.METHODSWe report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells.RESULTSThe cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD-). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion.CONCLUSIONSB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.TRIAL REGISTRATIONClinicalTrials.gov NCT03389035.FUNDINGThis study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).
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http://dx.doi.org/10.1172/JCI138473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598053PMC
November 2020

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

Mol Ther 2020 09 30;28(9):1974-1986. Epub 2020 May 30.

Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca/Fondazione MBBM, 20900 Monza, Italy.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.
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http://dx.doi.org/10.1016/j.ymthe.2020.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474266PMC
September 2020

Preclinical Efficacy and Safety of CD19CAR Cytokine-Induced Killer Cells Transfected with Sleeping Beauty Transposon for the Treatment of Acute Lymphoblastic Leukemia.

Hum Gene Ther 2018 05 16;29(5):602-613. Epub 2018 Apr 16.

1 Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca , San Gerardo Hospital/Fondazione MBBM, Monza, Italy.

Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expand on those findings by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with the Sleeping Beauty (SB) transposon CD19CAR (CARCIK-CD19). Specifically, thanks to a large-scale 18-day manufacturing process, it was possible to achieve stable CD19CAR expression (62.425 ± 6.399%) and efficient T-cell expansion (23.36 ± 3.00-fold). Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in an established patient-derived xenograft (PDX) of MLL-ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a biodistribution and toxicity model. The infused cells persisted in the hematopoietic and post-injection perfused organs until the end of the study and consisted of CD8, CD56, and CAR T cells. Overall, these findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL, a possibility that will be tested in Phase I/II clinical trials after allogeneic hematopoietic stem-cell transplantation.
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http://dx.doi.org/10.1089/hum.2017.207DOI Listing
May 2018

Redirecting T cells with Chimeric Antigen Receptor (CAR) for the treatment of childhood acute lymphoblastic leukemia.

J Autoimmun 2017 Dec 24;85:141-152. Epub 2017 Aug 24.

Centro Ricerca Tettamanti, Clinica Pediatrica, Università di Milano-Bicocca, Fondazione MBBM, Osp. San Gerardo, Monza, Italy.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Nowadays the survival rate is around 85%. Nevertheless, an urgent clinical need is still represented by primary refractory and relapsed patients who do not significantly benefit from standard approaches, including chemo-radiotherapy and hematopoietic stem cell transplantation (HSCT). For this reason, immunotherapy has so far represented a challenging novel treatment opportunity, including, as the most validated therapeutic options, cancer vaccines, donor-lymphocyte infusions and tumor-specific immune effector cells. More recently, unexpected positive clinical results in ALL have been achieved by application of gene-engineered chimeric antigen expressing (CAR) T cells. Several CAR designs across different trials have generated similar response rates, with Complete Response (CR) of 60-90% at 1 month and an Event-Free Survival (EFS) of 70% at 6 months. Relevant challenges anyway remain to be addressed, such as amelioration of technical, cost and feasibility aspects of cell and gene manipulation and the necessity to face the occurrence of relapse mechanisms. This review describes the state of the art of ALL immunotherapies, the novelties in terms of gene manipulation approaches and the problems emerged from early clinical studies. We describe and discuss the process of clinical translation, including the design of a cell manufacturing protocol, vector production and regulatory issues. Multiple antigen targeting and combination of CAR T cells with molecular targeted drugs have also been evaluated as latest strategies to prevail over immune-evasion.
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http://dx.doi.org/10.1016/j.jaut.2017.08.003DOI Listing
December 2017

Engineered T cells towards TNFRSF13C (BAFFR): a novel strategy to efficiently target B-cell acute lymphoblastic leukaemia.

Br J Haematol 2018 09 18;182(6):939-943. Epub 2017 Aug 18.

Centro Ricerca Tettamanti, Clinica Pediatrica, Università degli Studi di Milano-Bicocca, Osp. San Gerardo/Fondazione MBBM, Monza, Italy.

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http://dx.doi.org/10.1111/bjh.14899DOI Listing
September 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

TNFRSF13C (BAFFR) positive blasts persist after early treatment and at relapse in childhood B-cell precursor acute lymphoblastic leukaemia.

Br J Haematol 2018 08 1;182(3):434-436. Epub 2017 Jun 1.

Centro Ricerca Tettamanti, Clinica Pediatrica, Dipartimento di Medicina e Chirurgia, Università degli Studi di Milano-Bicocca, Fondazione MBBM, Monza, Italy.

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http://dx.doi.org/10.1111/bjh.14794DOI Listing
August 2018

Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia.

Mol Ther 2017 08 4;25(8):1933-1945. Epub 2017 May 4.

Istituto di Ricerca in Biomedicina, Università degli Studi della Svizzera Italiana, 6500 Bellinzona, Switzerland.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.
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http://dx.doi.org/10.1016/j.ymthe.2017.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542631PMC
August 2017

Acute Myeloid Leukemia Targeting by Chimeric Antigen Receptor T Cells: Bridging the Gap from Preclinical Modeling to Human Studies.

Hum Gene Ther 2017 03 1;28(3):231-241. Epub 2016 Dec 1.

1 Molecular Therapy Unit, Tettamanti Research Center, Pediatric Clinic, University of Milano Bicocca , San Gerardo Hospital/MBBM Foundation, Monza, Italy.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.
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http://dx.doi.org/10.1089/hum.2016.092DOI Listing
March 2017

Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform.

Oncotarget 2016 08;7(32):51581-51597

Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital/Fondazione MBBM, Monza, Italy.

Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy.
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http://dx.doi.org/10.18632/oncotarget.9955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239498PMC
August 2016

Development of advanced therapies in Italy: Management models and sustainability in six Italian cell factories.

Cytotherapy 2016 Apr;18(4):481-6

Laboratory of Cell and Gene Therapy Stefano Verri, San Gerardo Hospital, Monza, Italy; Tettamanti Research Center, Pediatric Clinic Monza Brianza per il Bambino e la sua Mamma (MBBM) Foundation, Monza, Italy.

On November 10, 2014, the representatives of all six certified Good Manufacturing Practices (GMP) cell factories operating in the Lombardy Region of Italy convened a 1-day workshop in Milan titled "Management Models for the Development And Sustainability of Cell Factories: Public-Private Partnership?" The speakers and panelists addressed not only the many scientific, technological and cultural challenges faced by Lombardy Cell Factories, but also the potential impact of advanced therapy medicinal products (ATMPs) on public health and the role played by translational research in this process. Future perspectives for research and development (R&D) and manufacturing processes in the field of regenerative medicine were discussed as well. This report summarizes the most important issues raised by the workshop participants with particular emphasis on strengths and limitations of the R&D and manufacturing processes for innovative therapeutics in Lombardy and what can be improved in this context while maintaining GMP standards. The participants highlighted several strategies to translate patient-specific advanced therapeutics into scaled manufacturing products for clinical application. These included (i) the development of a synergistic interaction between public and private institutions, (ii) better integration with Italian regulatory agencies and (iii) the creation of a network among Lombardy cell factories and other Italian and European institutions.
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http://dx.doi.org/10.1016/j.jcyt.2016.01.002DOI Listing
April 2016

Full GMP-compliant validation of bone marrow-derived human CD133(+) cells as advanced therapy medicinal product for refractory ischemic cardiomyopathy.

Biomed Res Int 2015 1;2015:473159. Epub 2015 Oct 1.

Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino, IRCCS, Via Parea 4, 20138 Milan, Italy ; Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Via Festa del Perdono 7, 20122 Milan, Italy.

According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).
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http://dx.doi.org/10.1155/2015/473159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606188PMC
August 2016

Donor-derived CD19-targeted T cells in allogeneic transplants.

Curr Opin Hematol 2015 Nov;22(6):497-502

aDepartment of Pediatrics, Tettamanti Research Center, University of Milano-Bicocca, Milan bSan Gerardo Hospital/Fondazione MBBM, Monza, Italy.

Purpose Of Review: Allogeneic hematopoietic stem cell transplantation (HSCT) is still partially ineffective in curing high-risk hematological malignancies, with estimates of relapse rates ranging from 40 to 50%. The purpose of this review is to discuss the emerging therapeutic options for patients with relapsed disease following HSCT based on adoptive immunotherapy using donor-derived T cells genetically engineered to express CD19-specific chimeric antigen receptors (CARs).

Recent Findings: Adoptive cell therapy (ACT) with CAR-modified T cells represents an attractive therapeutic option for further enhancing the graft-versus-leukemia effect. However, CAR-modified T cells are often obtained using apheresis products collected from the patient's own blood, a procedure that has hindered the application of CAR-based therapies into the clinic. Alternative approaches rely on CAR T cells derived from donors rather than the patient's own blood. Therefore, it appears that overcoming the practical limitation of allogeneic T cell-induced graft versus-host-disease is a key to providing access to CAR immunotherapy to all eligible patients.

Summary: Donor-derived CD19-CAR T cells may advance the field of CAR immunotherapy by controlling relapse in leukemic patients and improving the range of applications of ACT protocols.
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http://dx.doi.org/10.1097/MOH.0000000000000178DOI Listing
November 2015

Failure of interferon-γ pre-treated mesenchymal stem cell treatment in a patient with Crohn's disease.

World J Gastroenterol 2015 Apr;21(14):4379-84

Andrea Taddio, Alessandro Ventura, Institute for Maternal and Child Health - IRCCS "Burlo Garofolo" - Trieste and University of Trieste, 34100 Trieste, Italy.

Mesenchymal stem cells (MSC) are cells of stromal origin which exhibit unlimited self-renewal capacity and pluripotency in vitro. It has recently been observed that MSC may also exert a profound immunosuppressive and anti-inflammatory effect both in vitro and in vivo with consequent potential use in autoimmune disorders. We present the case of a patient suffering from childhood-onset, multidrug resistant and steroid-dependent Crohn's disease who underwent systemic infusions of MSC, which led to a temporary reduction in CCR4, CCR7 and CXCR4 expression by T-cells, and a temporary decrease in switched memory B-cells, In addition, following MSC infusion, lower doses of steroids were needed to inhibit proliferation of the patient's peripheral blood mononuclear cells. Despite these changes, no significant clinical benefit was observed, and the patient required rescue therapy with infliximab and subsequent autologous hematopoietic stem cell transplantation. The results of biological and in vitro observations after MSC use and the clinical effects of infusion are discussed, and a brief description is provided of previous data on MSC-based therapy in autoimmune disorders.
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http://dx.doi.org/10.3748/wjg.v21.i14.4379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394101PMC
April 2015

CD123 AML targeting by chimeric antigen receptors: A novel magic bullet for AML therapeutics?

Oncoimmunology 2014;3:e28835. Epub 2014 May 14.

Cancer Research UK; London Research Institute; Haematopoietic Stem Cell Laboratory; London, UK.

Chimeric antigen receptor (CAR) modified T cells have emerged as powerful tools for controlling leukemias. We recently showed that anti-CD123 CAR-expressing cytokine-induced killer T cell treatment is an effective immunotherapeutic approach to eradicate Acute Myeloid Leukemia (AML) cells. Here, we discuss how this genetically modified cell-based strategy could be relevant to the field of AML therapeutics.
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http://dx.doi.org/10.4161/onci.28835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106165PMC
May 2014

Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media.

Stem Cell Res Ther 2014 Jan 9;5(1). Epub 2014 Jan 9.

Introduction: Despite having a proven immunosuppressive potential in vitro, human mesenchymal stromal cells (MSCs) are reported to display variable efficacy in vivo and, in fact, their proven benefit in the clinical practice is still limited and controversial.

Methods: The interplay between clinical grade MSCs and pre-activated donor lymphocytes or selected lymphocyte subsets was studied in vitro. The kinetics of MSC growth and viability was evaluated by adhesion-dependent changes of culture plate impedance and biochemically by a colorimetric assay. Activation of natural killer (NK) cells was assessed as well, using a flow cytometry assay.

Results: A strong inhibition of MSC growth was rapidly induced by the addition of pre-activated lymphocytes but not of resting lymphocytes. Inhibition seems not to be attributable to a single cell population, as similar results can be obtained by depleting NK cells or by using either selected CD4+ or CD8+ lymphocytes. In addition, conditioned medium (CM) from activated lymphocytes was able to inhibit MSC growth in a dose-dependent manner. Furthermore, licensing with IFN-γ partially protected MSCs from pre-activated lymphocytes but not from their CM. These results suggest an inhibitory role of lymphocyte-activation-derived substances. However, the identification of a single molecule responsible for MSC inhibition remained elusive, even if preliminary experiments showed that ATP and, to a lesser extent, TNF-α might play a role.

Conclusions: These results suggest that survival of MSCs can be affected by soluble mediators released by activated lymphocytes. Thus it can be hypothesized that MSC immunosuppressive action in vivo could be impaired by ongoing immune activation through the release of inflammatory mediators.
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http://dx.doi.org/10.1186/scrt392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055165PMC
January 2014

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

Biol Blood Marrow Transplant 2014 Mar 7;20(3):375-81. Epub 2013 Dec 7.

HSCT Pediatric Unit and Laboratory of Cell Therapy "S. Verri", San Gerardo Hospital, Monza, Italy.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.
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http://dx.doi.org/10.1016/j.bbmt.2013.11.033DOI Listing
March 2014

Immunosuppression does not affect human bone marrow mesenchymal stromal cell efficacy after transplantation in traumatized mice brain.

Neuropharmacology 2014 Apr 15;79:119-26. Epub 2013 Nov 15.

IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Department of Neuroscience, Milan, Italy.

The need for immunosuppression after allo/xenogenic mesenchymal stromal cell (MSC) transplantation is debated. This study compared the long-term effects of human (h) bone marrow MSC transplant in immunocompetent or immunosuppressed traumatic brain injured (TBI) mice. C57Bl/6 male mice were subjected to TBI or sham surgery followed 24 h later by an intracerebroventricular infusion of phosphate buffer saline (PBS, control) or hMSC (150,000/5 μl). Immunocompetent and cyclosporin A immunosuppressed (CsA) mice were analyzed for gene expression at 72 h, functional deficits and histological analysis at five weeks. Gene expression analysis showed the effectiveness of immunosuppression (INFγ reduction in CsA treated groups), with no evidence of early rejection (no changes of MHCII and CD86 in all TBI groups) and selective induction of T-reg (increase of Foxp3) only in the TBI hMSC group. Five weeks after TBI, hMSC had comparable efficacy, with functional recovery (on both sensorimotor and cognitive deficits) and structural protection (contusion volume, vessel rescue effect, gliotic scar reduction, induction of neurogenesis) in immunosuppressed and immunocompetent mice. Therefore, long-term hMSC efficacy in TBI is not dependent on immunosuppressive treatment. These findings could have important clinical implication since immunosuppression in acute TBI patients may increase their risk of infection and not be tolerated.
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http://dx.doi.org/10.1016/j.neuropharm.2013.11.001DOI Listing
April 2014

Allogeneic mesenchymal stem cell infusion for the stabilization of focal segmental glomerulosclerosis.

Biologicals 2013 Nov 14;41(6):439-45. Epub 2013 Oct 14.

Nephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy.

Focal segmental glomerulosclerosis (FSGS) is the most frequent acquired renal condition resulting in end stage kidney disease in children. We describe a cell therapy treatment with human allogeneic bone marrow mesenchymal stem cells (MSC) in a 13-year-old patient developing recurrent FSGS after renal transplantation, which was not responding to conventional therapy. This treatment relied on the following measurements:clinical and laboratory evaluation of renal function, proteome array, biopsy, short tandem repeat assay. Before MSC treatment, the patient needed weekly plasmapheresis to achieve proteinuria-to-creatininuria ratio below 5. After three MSC infusions without adverse events, the patient has a stable renal function and the proteinuria target was reached without plasmapheresis. In addition, some circulating inflammatory factors decreased and their levels were still low after one year. This is the first report of an MSC treatment in an FSGS patient. Even though different factors may have contributed to the clinical results, after MSC infusion a stable reduction in the serum level of several inflammatory factors has been registered and the patient does not need anymore plasmapheresis to keep proteinuria under control. In addition, this encouraging single case let us identify some putative efficacy biomarkers that could be of clinical interest in chronic kidney diseases.
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http://dx.doi.org/10.1016/j.biologicals.2013.09.004DOI Listing
November 2013

Acute myeloid leukemia and novel biological treatments: monoclonal antibodies and cell-based gene-modified immune effectors.

Immunol Lett 2013 Sep-Oct;155(1-2):43-6. Epub 2013 Sep 25.

Centro di Ricerca Matilde Tettamanti, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy.

In the context of acute myeloid leukemia (AML) treatment, the interface between chemotherapy and immunotherapy is at present getting closer as never before. Scientific research is oriented in overcoming the main limits of actual chemotherapeutic regimens against AML, which still accounts for a considerable number of relapsed or resistant forms. A lot of investments have been done in the use of monoclonal antibodies (mAbs) and recently gene-modified immune cells have been considered as an alternative approach whenever chemotherapy fails to eradicate the disease. In this sense, AML is a potential suitable target for immunotherapeutic approaches, due to overexpression of several tumor antigens. Here we describe the state of the art of mAbs and cellular therapies employing engineered immune effectors, developed against specific AML antigens, in a window embracing preclinical research and translational studies to the clinical setting.
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http://dx.doi.org/10.1016/j.imlet.2013.09.013DOI Listing
July 2014

Advanced targeted, cell and gene-therapy approaches for pediatric hematological malignancies: results and future perspectives.

Front Oncol 2013 30;3:106. Epub 2013 Apr 30.

Department of Pediatrics, Centro di Ricerca Matilde Tettamanti, San Gerardo Hospital, University of Milano-Bicocca Monza, Italy.

Despite the survival of pediatric patients affected by hematological malignancies being improved in the last 20 years by chemotherapy and hematopoietic stem cell transplantation, a significant amount of patients still relapses. Treatment intensification is limited by toxic side effects and is constrained by the plateau of efficacy, while the pipeline of new chemotherapeutic drugs is running short. Therefore, novel therapeutic strategies are essential and researchers around the world are testing in clinical trials immune and gene-therapy approaches as second-line treatments. The aim of this review is to give a glance at these novel promising strategies of advanced medicine in the field of pediatric leukemias. Results from clinical protocols using new targeted "smart" drugs, immunotherapy, and gene therapy are summarized, and important considerations regarding the combination of these novel approaches with standard treatments to promote safe and long-term cure are discussed.
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http://dx.doi.org/10.3389/fonc.2013.00106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639447PMC
May 2013

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

J Immunother 2013 Apr;36(3):181-9

Etablissement Français du Sang, 29 av du Maquis du Grésivaudan, BP35, 38701 La Tronche, France.

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.
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http://dx.doi.org/10.1097/CJI.0b013e318288f8c1DOI Listing
April 2013

Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor.

Br J Haematol 2013 May 25;161(3):389-401. Epub 2013 Feb 25.

Centro di Ricerca Matilde Tettamanti, Department of Paediatrics, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy.

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T-cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34(+) leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine-induced killer (CIK) cells were transduced with SFG-retroviral-vector encoding an anti-CD123 CAR. Transduced cells were able to strongly kill CD123(+) cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti-CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti-CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123-low-expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti-CD123.CAR. Taken together, our results indicate that CD123-specific CARs strongly enhance anti-AML CIK functions, while sparing HSPCs and normal low-expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.
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http://dx.doi.org/10.1111/bjh.12282DOI Listing
May 2013

Mesenchymal stem/stromal cells: a new ''cells as drugs'' paradigm. Efficacy and critical aspects in cell therapy.

Curr Pharm Des 2013 ;19(13):2459-73

Laboratorio di Biotecnologie applicate all'Ortopedia, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788322PMC
http://dx.doi.org/10.2174/1381612811319130015DOI Listing
September 2013

Mesenchymal stem/stromal cells: a new ''cells as drugs'' paradigm. Efficacy and critical aspects in cell therapy.

Curr Pharm Des 2013 ;19(13):2459-73

Laboratorio di Biotecnologie applicate all'Ortopedia, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788322PMC
http://dx.doi.org/10.2174/1381612811319130015DOI Listing
September 2013

Basic biology and clinical application of multipotent mesenchymal stromal cells: from bench to bedside.

Stem Cells Int 2012 28;2012:185943. Epub 2012 Aug 28.

Departments of Hematology and Oncology, University Children's Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.

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http://dx.doi.org/10.1155/2012/185943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434412PMC
September 2012

From bone marrow transplantation to cellular therapies: possible therapeutic strategies in managing autoimmune disorders.

Curr Pharm Des 2012 ;18(35):5776-81

Institute for Maternal and Child Health – IRCCS Burlo Garofolo, Trieste, Italy.

Chronic inflammatory disorders occurring in childhood represent a serious therapeutic challenge. However, available therapies seem not to be targeted on the pathogenic mechanism of the disease and are often not actively affecting the natural history of the disease. Emerging treatments might be of some benefit to many patients who did not respond to conventional therapeutic options. Biological therapies with monoclonal antibodies and other recombinant proteins have been introduced in clinical practice. At the same time, mesenchymal stromal cells (MSC) have gained attention as a savage treatment in patients subjected to hematopoietic stem cell transplantation who develop severe graft versus host disease (GvHD); in addition, recent reports from clinical trials on larger cohorts of patients support their use as second-line treatment after failure of corticosteroid treatment. For analogy, they have been proposed for the treatment of intractable autoimmune disorders. Hematopoietic stem cell transplantation (HSCT) has been shown to be effective for treatment of rheumatic disorder cases that were resistant to traditional therapies especially if combined with cell manipulation techniques, such as selection of regulatory T cell and depletion of harmful lymphocytes. We herein present the rationale of different strategies, the preliminary data obtained in clinical trials, unsolved problems and possible next developments of novel treatment protocols of autoimmune disorders.
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http://dx.doi.org/10.2174/138161212803530754DOI Listing
May 2013

Mesenchymal stromal cells do not increase the risk of viral reactivation nor the severity of viral events in recipients of allogeneic stem cell transplantation.

Stem Cells Int 2012 30;2012:690236. Epub 2012 May 30.

Clinica Pediatrica, Università degli Studi di Milano Bicocca, Ospedale San Gerardo, 20900 Monza, Italy.

Mesenchymal stromal cells (MSC) are tested in clinical trials to treat graft versus host disease (GvHD) after stem cell transplantation (SCT). In vitro studies demonstrated MSC's broad immunosuppressive activity. As infections represent a major risk after SCT, it is important to understand the role of MSC in this context. We analyzed 24 patients (pts) receiving MSC for GvHD in our Unit between 2009 and 2011. We recorded viral reactivations as measured in whole blood with polymerase chain reaction for 100 days following MSC administration. In patients with a documented viral reactivation in the first 3 days following MSCs infusion the frequency of virus-specific IFNgamma-producing cells was determined through enzyme-linked immunospot assay. In our cohort of patients viral reactivation after MSC infusion occurred in 45% of the cases, which did not significantly differ from the incidence in a historical cohort of patients affected by steroid resistant GvHD and treated with conventional immunosuppression. No patient presented severe form of infection. Two cases could be checked for immunological response to viral stimulus and demonstrated virus specific T-cytotoxic lymphocyte activity. In our experience MSC infusion did not prove to trigger more frequent or severer viral reactivations in the post transplantation setting.
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http://dx.doi.org/10.1155/2012/690236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369546PMC
August 2012

New advances in leukaemia immunotherapy by the use of Chimeric Artificial Antigen Receptors (CARs): state of the art and perspectives for the near future.

Ital J Pediatr 2011 Sep 22;37:46. Epub 2011 Sep 22.

Centro di Ricerca Fondazione "Matilde Tettamanti", Clinica Pediatrica Azienda Ospedaliera San Gerardo, Università Milano-Bicocca, Monza, Italy.

Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs), which are artificial molecules containing antibody-derived fragments (to bind the specific target), joined with potent signalling T-Cell Receptor (TCR)-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future.
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http://dx.doi.org/10.1186/1824-7288-37-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195094PMC
September 2011

Comparison of two CD40-ligand/interleukin-2 vaccines in patients with chronic lymphocytic leukemia.

Cytotherapy 2011 Oct 12;13(9):1128-39. Epub 2011 Jul 12.

Center for Cell and Gene Therapy, Baylor College of Medicine, The Methodist Hospital and Texas Children's Hospital, Houston, TX 77030, USA.

Background Aims: Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies.

Methods: We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity.

Results: We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine.

Conclusions: CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients.
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http://dx.doi.org/10.3109/14653249.2011.592523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3687103PMC
October 2011