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Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Summary Background Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, have been proposed as a promising therapeutic option for people with multiple sclerosis on the basis of their immunomodulatory and neuroprotective properties. The MEsenchymal StEm cells for Multiple Sclerosis (MESEMS) study was devised to evaluate the safety, tolerability, and activity of autologous MSCs derived from bone marrow and infused intravenously in patients with active multiple sclerosis. Methods MESEMS is a randomised phase 2 trial done at 15 sites in nine countries. Patients (aged 18–50 years) with active relapsing-remitting or progressive multiple sclerosis were included if they had a disease duration of 2–15 years since onset of multiple sclerosis and an Expanded Disability Status Scale score of 2·5–6·5. Patients were randomly assigned (1:1), according to a crossover design, to receive a single intravenous dose of autologous bone marrow-derived MSCs followed by placebo at week 24, or to receive placebo followed by autologous MSCs at week 24, with a follow-up visit at week 48. Primary objectives were to test safety and activity of MSC treatment. The primary safety endpoint was to assess the number and severity of adverse events within each treatment arm. The primary efficacy endpoint was the number of gadolinium-enhancing lesions (GELs) counted over week 4, 12, and 24 compared between treatment groups. The primary efficacy endpoint was assessed in the full analyis set, after all participants completed the week 24 visit. Efficacy endpoints were evaluated using a predefined statistical testing procedure. Safety was monitored throughout the study by recording vital signs and adverse events at each visit. Findings From July 16, 2012, until July 31, 2019, 144 patients were randomly assigned to first receive early intravenous infusion of autologous bone marrow-derived MSCs (n=69) or placebo (n=75). MSC treatment did not meet the primary endpoint of efficacy on the total number of GELs accumulated from baseline to week 24 (rate ratio [RR] 0·94, 95% CI 0·58–1·50; p=0·78). 213 adverse events were recorded, similarly distributed between groups (93 cases recorded in 35 [51%] of 69 patients treated first with MSCs vs 120 cases in 42 [56%] of 75 patients infused first with placebo). The most frequent adverse events reported were infection and infestations, with a total of 54 (25%) of 213 adverse events (18 [19%] of 93 in the early-MSC group and 36 [30%] of 120 in the delayed-MSC group). Nine serious adverse events were reported in seven patients treated with placebo versus none in the MSC group. All serious adverse events were considered to be unrelated to the treatment infusion. No deaths were reported during the study. Interpretation Bone marrow-derived MSC treatment was safe and well tolerated but did not show an effect on GELs, an MRI surrogate marker of acute inflammation, in patients with active forms of multiple sclerosis, at week 24. Thus, this study does not support the use of bone marrow-derived MSCs to treat active multiple sclerosis. Further studies should address the effect of MSCs on parameters related to tissue repair. Funding Fondazione Italiana Sclerosi Multipla (FISM), the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS), and the Multiple Sclerosis International Federation (MSIF) for centralised activities. Individual trials participating in the MESEMS network are funded by the following agencies: FISM and Compagnia di San Paolo (Italy); The Danish Multiple Sclerosis Society, The Toyota Foundation, and Danish Blood Donors’ Research Foundation (Denmark); the Spanish Health Research Institute Carlos 3 and the Andalusian Public Foundation Progreso y Salud (Spain); the Royan Institute for Stem Cell Biology and Technology (Iran); the Spinal Cord Injury and Tissue Regeneration Centre Salzburg, Paracelsus Medical University, and Salzburg (Austria); the Fondation pour l’aide a la recherche sur la sclerose en plaques (ARSEP), French Muscular Dystrophy Association (AFM)-Telethon (France); the UK Multiple Sclerosis Society and the UK Stem Cell Foundation (UK); and the Multiple Sclerosis Society of Canada and The Multiple Sclerosis Scientific Research Foundation and Research Manitoba (Canada).

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pre-transplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary end-point was the safety profile of MSC administration during the one-year follow-up. Nineteen patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56bright NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pre-transplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.

BACKGROUND: Chimeric 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. METHODS: We 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. RESULTS: The 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. CONCLUSION: SB-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 REGISTRATION: ClinicalTrials.gov NCT03389035. FUNDING: This 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).

Here we report the case of successful immune tolerance induction in a living‐donor kidney transplant recipient remotely treated with autologous bone marrow‐derived mesenchymal stromal cells (MSC). This case report, which to the best of our knowledge is the first in the world in this setting, provides evidence that the modulation of the host immune system with MSC can enable the safe withdrawal of maintenance immunosuppressive drugs while preserving optimal long‐term kidney allograft function., This report provides the evidence that autologous BM‐MSC infusion induces a pro‐tolerogenic immune environment, thus allowing complete discontinuation of antirejection drugs in kidney transplantation

Breast cancer is the most common malignancy among women worldwide, and HER2-positive breast cancer accounts for approximately 15% of all breast cancer diagnoses. The advent of HER2-targeting therapies has dramatically improved the survival of these patients, significantly reducing their risk of recurrence and death. However, as a significant proportion of patients ultimately develop resistance to these therapies, it is extremely important to identify new treatments to further improve their clinical outcomes. Immunotherapy has revolutionized the treatment and history of several cancer types, and it has already been approved as a standard of care for patients with triple-negative breast cancer. Based on a strong preclinical rationale, immunotherapy in HER2-positive breast cancer represents an intriguing field that is currently under clinical investigation. There is a close interplay between HER2-targeting therapies (both approved and under investigation) and the immune system, and several new immunotherapeutic strategies, including immune checkpoint inhibitors, CAR-T cells and therapeutic vaccines, are being studied in this disease. In this narrative review, we discuss the clinical evidence and the future perspectives of immunotherapy for patients with HER2-positive breast cancer.

Background aims: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. Methods: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy “Plagencell network” for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. Results: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at

Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic option for limiting chronic kidney disease progression. Conditioned medium (CM) containing bioactive compounds could convey similar benefits, avoiding the potential risks of cell therapy. This study compared the efficacy of nonrenal and renal cell-based therapy with the corresponding CM in rats with renal mass reduction (RMR). Infusions of human kidney stromal cells (kPSCs) and CM-kPSCs, but not umbilical cord (uc) MSCs or CM-ucMSCs, reduced proteinuria and preserved podocyte number and nephrin expression in RMR rats. Glomerular fibrosis, microvascular rarefaction, and apoptosis were reduced by all treatments, while the peritubular microvascular loss was reduced by kPSCs and CM-kPSCs treatment only. Importantly, kPSCs and CM-kPSCs reduced NG2-positive pericytes, and all therapies reduced α-smooth muscle actin expression, indicating reduced myofibroblast expansion. Treatment with kPSCs also significantly inhibited the accumulation of ED1-positive macrophages in the renal interstitium of RMR rats. These findings demonstrate that the CM of ucMSCs and kPSCs confers similar renoprotection as the cells. kPSCs and CM-kPSCs may be superior in attenuating chronic renal injury as a cell source.

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.

Introduction Allogeneic Chimeric Antigen Receptor (CAR) T cells engineered with non-viral methods offer a modality to reduce costs and logistical complexity of the viral process and allow lymphodepleted patients to access CAR T cell treatment. We recently proposed the use of Sleeping Beauty (SB) transposon to engineer donor-derived T cells differentiated according to the cytokine-induced killer (CIK) cell protocol (Magnani CF et al. J Clin Invest. 2021). We report here outcomes on B-cell acute lymphoblastic leukemia (B-ALL) patients, relapsing after transplantation, treated with donor-derived anti-CD19 CAR T cells (CARCIK-CD19). Methods We conducted an academic, multi-center, phase I/II dose-escalation trial in patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The infusion product was manufactured in-house starting from 50 mL of peripheral blood from the HSCT donor by electroporation with GMP-grade plasmids. All patients underwent lymphodepletion with Fludarabine (30 mg/m 2/day x 4 days) and Cyclophosphamide (500 mg/m 2/day x 2 days), before proceeding to CARCIK-CD19 infusion. We used the Bayesian Optimal Interval (BOIN) design to define a four-dose escalation scheme. Primary objectives were to define the Maximum Tolerated Dose (MTD), safety, and feasibility. Secondary objectives included the assessment of complete hematologic response (CR), duration of response (DOR), progression-free (PFS), event-free (EFS), and overall survival (OS). This study was registered at ClinicalTrials.gov, NCT03389035. Results From January 2018 to June 2021, a total of 32 patients were screened, 26 enrolled (6 children and 20 adults) and 21 infused (4 children and 17 adults). Reasons for not receiving infusion included consent withdrawal (N=1), disease progression not controlled by bridging therapy (N=3), acquisition of myeloid phenotype (N=1). The median number of prior therapies was 4 (range, 1-7) with a median time interval from HSCT to relapse of 9 months. The median BM blasts was 60% (range, 5-100%) at enrollment and 7% (range, 0-96%) post lymphodepletion. Of the 21 patients infused, CARCIK-CD19 were obtained by HLA-identical sibling (n=6, 29%), matched unrelated (n= 7, 33%), and haploidentical donors (n=8, 38%). Three patients (14%) received the first dose level of 1x10 6 CARCIK-CD19 cells/Kg, three (14%) the second of 3x10 6, and three (14%) the third of 7.5x10 6 whereas 12 patients (57%) received the fourth and last planned dose level of 15x10 6 cells/Kg, as no dose limiting toxicity (DLT) was observed. CRS was observed in six patients (three grade I and three grade II) and immune effector cell-associated neurotoxicity in two patients at the highest dose. Although 9 out of 21 had experienced acute or chronic graft-versus-host disease (GvHD) after the previous HSCT, secondary GvHD was never induced by CARCIK-CD19. Complete response was achieved by 13 out of 21 patients (61.9%, 95%CI=38-82%) and by 11 out of 15 patients treated with the 2 highest doses (73.3%, 95%CI=45-92%). Eleven of these responders were MRD-negative. Notably, the type of donor did not influence the achievement of CR 28 days post-infusion. At a median follow up of 21.6 months (range, 1.0-38.4 months), 10 patients (47.6%) are alive in CR (9 in the 2 highest dose levels). Overall, the median OS and EFS were 9.7 and 3.2 months, respectively, with a median DOR of 4.0 months (range, 1.0-23.5 months). Patients in CR at 28-days had a 6-months relapse-free survival of 48.4% (SE=14.9). EFS at 6 months was 26.5% (SE=9.9) and OS was 67.6% (SE=11.1). Among the 13 patients who achieved CR, two children underwent consolidation with a second allo-HSCT in complete remission. Adult patients did not receive any additional anti-leukemic therapies unless a relapse occurred, and four of them remained in remission and alive (+24, +9, +6, and +4 months). Robust CARCIK-CD19 cell expansion was achieved in most patients and CARCIK-CD19 cells were measurable for up to 22 months. Conclusions SB-engineered CAR T cells induce sustained responses in B-ALL patients relapsed after HSCT irrespective of the donor type and without severe toxicities. Disclosures Lussana: Incyte: Honoraria; Pfizer: Honoraria; Astellas Pharma: Honoraria; Amgen: Honoraria. Gritti: Takeda: Consultancy; Roche: Consultancy; Kite Gilead: Consultancy; IQvia: Consultancy; Italfarmaco: Consultancy; Clinigen: Consultancy. Biondi: Incyte: Consultancy, Other: Advisory Board; Bluebird: Other: Advisory Board; Novartis: Honoraria; Amgen: Honoraria; Colmmune: Honoraria.

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 invitro and invivo 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.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Background Significant efforts over the past few years led Chimeric Antigen Receptor (CAR) T cell therapy to success in relapsed and refractory (r/r) B-cell malignancies. Still logistical complexity, high costs and toxicities are currently the main barriers to the use of CAR T cell therapy. We therefore propose non-viral engineering of an allogeneic T cell population according to cytokine induced killer (CIK) cell protocol of differentiation. Methods We reported the updated results of our 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 CIK (CARCIK-CD19) according to the method enclosed in the filed patent EP20140192371. After lymphodepletion with Fludarabine (30 mg/m2/day) x 4 days and Cyclophosphamide (500 mg/m2/day) x 2 days, CARCIK-CD19 were infused following a four-dose escalation scheme (1x106, 3x106, 7.5x106 and 15x106 transduced CAR+ T cells/kg) according to the Bayesian Optimal Interval Design (BOIN). During the cell manufacturing period, bridging anti leukemic therapy from patient registration to the beginning of the lymphodepletion, was allowed. The primary endpoint was to define the Maximum Tolerated Dose (MTD) and the safety assessment. Key secondary endpoints included the assessment of complete hematologic response (CR), defined as < 5% bone marrow (BM) blasts, circulating blasts < 1%, no clinical evidence of extramedullary disease, as well as the characterization of CARCIK-CD19 persistence in PB and BM (NCT03389035). Results The cellular product was produced successfully for all patients starting from the donor-derived peripheral blood (PB) and consisted mostly of CD3+ lymphocytes (mean 98.85% ±SD 1.19%) with a mean of 38.6% CAR expression (range 15.10%-73.17%). From January 2018 to July 2020, a total of 24 patients were screened, and 15 were enrolled (4 children and 11 adults) and infused with a single dose of CARCIK-CD19 (n=3 HLA identical sibling, n=4 MUD, n=8 haploidentical donor). The leukemic burden in the BM post lymphodepletion/pre-infusion ranged from 0% to 96%. Robust expansion was achieved in the majority of the patients. The maximal expansion reached about 1x106 transgene copies per μg DNA and 70% of CAR+ T cells in PB. CD8+ T cells represented the predominant circulating CAR+ T cell subset. Persistence of central memory CAR+ T cells was observed after infusion and CAR T cells were measurable up to 9 months. CARCIK-CD19 were characterized by a high profile of safety in all treated patients. Toxicities reported were two grade I and two 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. Seven out of 9 patients, receiving the highest doses, achieved CR and CRi at day 28. MRD-negative status for all responders was achieved by 6 out of 9 patients (1 currently in evaluation). The two patients in CR but with MRD+ relapsed with a CD19+ disease at +2.3 and +1.9 months post infusion, respectively. Among the 6 patients who achieved MRD-negative CR, two children underwent consolidation with a second allo-HSCT and are still alive and disease free (+17 and +13 months), two adult patients died of subsequent CD19+ disease relapse and two adult patients are still alive and disease free (+14 and +12 months) without additional therapies. The distribution profile of integration sites (IS) showed no preference for gene dense or promoter regions, and no particular differences between pre- and post- infusion sample IS. Samples harvested at early time points after infusion showed a highly polyclonal repertoire. At later time points (≥ 28 days after infusion) the repertoire of IS showed a marked reduction towards oligoclonality, in absence of specific dominant clones. Conclusions We can conclude that SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Sustained response was achieved without severe toxicities. All analyzed samples appear to have a highly polyclonal IS repertoire and no signs of genotoxicity by transposon insertions could be observed. Disclosures Gritti: IQVIA: Consultancy; Amgen: Honoraria; Autolus: Consultancy; Italfarmaco: Consultancy; F. Hoffmann-La Roche Ltd: Honoraria; Jannsen: Other: Travel Support; Takeda: Honoraria; Kite: Consultancy. Rambaldi:Sanofi: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Omeros: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Research grant from Amgen Inc.; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Advisory board and speaker fees from Pfizer.; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support from Gilead.; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support of parent study and funding of editorial support. Received travel support., Research Funding; University of Milan: Current Employment; BMS/Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Astellas: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company).

Highlights • Seventeen EBMT centers participated to a questionnaire on MSC manufacturing. • 88% of centers manufacture MSC from bone marrow and only 2 centers from umbilical cord. • Human platelet lysate has replaced bovine serum as culture medium supplement. • Release criteria extensively differ among centers. • The results highlight the need to harmonize MSC manufacturing., The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients’ stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

Background Mesenchymal stromal cell (MSC)-based therapy is a promising strategy for preventing the progression of chronic kidney disease (CKD), with the potential to induce tissue regeneration. In search of the best cellular source we compared, in the rat model of adriamycin (ADR) nephropathy, the regenerative potential of human stromal cells of non-renal origin, such as bone marrow (bm) MSCs and umbilical cord (uc) MSCs, with that of newly discovered stromal cells of renal origin, the kidney perivascular cells (kPSCs) known to exhibit tissue-specific properties. Methods The therapeutic effect of repeated infusions of human bmMSCs, ucMSCs, kPSCs (1.5 × 106 cells/rats) or conditioned medium from ucMSCs was studied in athymic rats with ADR-induced nephropathy (7.9 mg/kg). The ability of the three stromal cell populations to engraft the damaged kidney was evaluated by detecting the presence of human nuclear antigenpos cells. Glomerular podocyte loss and endothelial damage, sclerotic lesions and inflammation were assessed at 14 and 28 days. In-vitro experiments with a transwell system were performed to investigate the effects of different stromal cell populations on parietal epithelial cells (PECs) activated or not with albumin or angiotensin II for 24 h. Results Infusions of non-renal and renal stromal cells resulted in a comparable engraftment into the kidney, in the peritubular areas and around the glomerular structures. All three cell populations limited podocyte loss and glomerular endothelial cell injury, and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis. Human ucMSCs had an anti-inflammatory effect superior to that of the other stromal cells, reducing macrophage infiltration and inducing polarisation towards the M2 macrophage phenotype. Conditioned medium from ucMSCs shared the same renoprotective effects of the cells. Consistent with in-vivo data, bmMSCs and kPSCs, but even more so ucMSCs, limited proliferation, migratory potential and extracellular matrix production of activated PECs, when cultured in a transwell system. Conclusions Our data indicate that either non-renal or renal stromal cells induce renal tissue repair, highlighting ucMSCs and their conditioned medium as the most reliable clinical therapeutic tool for CKD patients. Electronic supplementary material The online version of this article (10.1186/s13287-018-0960-8) contains supplementary material, which is available to authorized users.

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab′)2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.

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% CD3+CD56+ 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 naive CCR7+CD45RA+ 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 × 106 CIK/kg. Discussion We conclude that CB-CIKs, combined with bispecific T-cell–engaging antibodies, offer a novel, effective treatment strategy for leukemia.

Background Immunotherapy using patient-derived CAR T cells has achieved complete remission and durable response in highly refractory populations. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Allogeneic Cytokine Induced Killer (CIK) cells, a T-cell population characterized by the enrichment of CD3+CD56+ cells, have demonstrated a high profile of safety in acute lymphoblastic leukemia (ALL) patients (Introna M et al. Biol Blood Marrow Transplant. 2017). CIK cells could be easily engineered by the non-viral Sleeping Beauty (SB) transposon for the clinical application (Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017). Methods CIK cells were generated from 50 ml of donor-derived peripheral blood (PB) by electroporation with the GMP-grade CD19.CAR/pTMNDU3 and pCMV-SB11 plasmids according to the method enclosed in the filed patent EP20140192371. After lymphodepletion with Fludarabine (30 mg/m2/day) x 4 days and Cyclophosphamide (300 mg/m2/day) x 2 days, CARCIK-CD19 were infused in pediatric and adult B-cell ALL (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The clinical trial follows a four-dose escalation scheme (1x106, 3x106, 7.5x106 and 15x106 transduced CAR+ T cells/kg) using the novel Bayesian Optimal Interval Design (BOIN). During the cell manufacturing period, bridging anti leukemic therapy from patient registration to the beginning of the lymphodepletion, was allowed. The primary endpoint was to define the Maximum Tolerated Dose (MTD) and a safety assessment. Key secondary endpoints included the assessment of complete hematologic response (CR), defined as < 5% bone marrow (BM) blasts, circulating blasts < 1%, no clinical evidence of extramedullary disease, as well as the characterization of CARCIK-CD19 persistence in PB and BM (NCT03389035). Results We manufactured eighteen batches by seeding a median of 126.8x106 allogeneicPBMCs. At the end of expansion, the mean harvesting was 6.46x109 nucleated cells (range 1.39 - 16.00x109). Manufactured cells were mostly CD3+ lymphocytes (mean 98.90% ±SE 0.30%). Of these, 43.57%±3.73% were CAR+, 47.07%±2.74% were CD56+, 80.44%±2.53% were CD8+. The quality requirements for batch release were met in 17 productions. As of the data cut-off date (July 19, 2019), 4 pediatric and 7 adult patients were infused with a single dose of CARCIK-CD19 (n=2 HLA identical sibling, n=4 MUD, n=5 haploidentical donor). The leukemic burden in the BM post lymphodepletion/pre-infusion ranged from 0% to 96%. CARCIK-CD19 were characterized by a high profile of safety in all treated patients. Toxicities reported were a grade I cytokine release syndrome and an infusion-related DMSO-associated seizure, with absence of dose-limiting toxicities, neurotoxicity and graft-versus-host disease (GvHD) in any of the treated patients. Four out of 5 patients, receiving the highest doses, achieved CR and CRi at day 28. The 3 patients in CR were also MRD- (by flow cytometry and RT-PCR) while the CRi was MRD+ and relapsed at day+49. Robust expansion was achieved in the majority of the patients as defined by detectable CAR T-cell detection (vector copy number VCN, range 4645-977992 transgene copies/ug) and flow (range 0.5-30%) in PB. The median time to peak engraftment was 14 days. The magnitude of expansion was correlated with the CD19+ burden in the BM at the time of the infusion (P value = 0.0006, R square 0.7469). CD8+ T cells represented the predominant CARCIK-CD19 T-cell subset (78.88%±5.33% d14 n=6) along with CD3+CD56+ CIK cells and CD4+ T cells to a lesser extent. The majority of CAR T cells had a central and effector memory phenotype. CAR T cells were measurable by VCN up to 6 months with a mean persistence of 70.5 ± 14.85 days (follow up ranging from 28 days to 1 year). No major difference was observed by integration analyses of the patients' PB and the cell products. The vector integration sites reflected the classical random distribution of SB without any tendency for gene dense regions. Conclusions Our ongoing phase I/II trial demonstrates that SB-engineered CARCIK-CD19 cells are able to expand and persist in pediatric and adult B-ALL patients relapsed after HSCT, with important implications for a non-viral technology. These encouraging results prompted us to expand our study. Disclosures Gritti: Autolus Ltd: Honoraria; Roche: Other: Not stated; Abbvie: Other: Not stated; Becton Dickinson: Other: Not stated. Rambaldi:Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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 × 106/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.

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 × 10 6 CD34+ cells/kg, and 3.4% collected 6 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 6 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.

Background Obinutuzumab (GA101) is a third generation, glycoengineered defucosylated anti-CD20 antibody which shows higher binding affinity for CD16A than fully glycosylated rituximab (RTX). This higher binding leads to stronger NK mediated antibody dependent cellular cytotoxicity activity (ADCC) by obinutuzumab compared to that induced by RTX. The GPI-anchored CD16B molecule is highly homologous to CD16A and is the major FcγR on polymorphonuclear neutrophils (PMN). We have therefore investigated the binding of obinutuzumab to CD16B and its functional activity on human PMN compared to parent rituximab (RTX) or to defucosylated rituximab (G2 antibody). Methods Binding to CD16B of glycoengineered or fully glycosylated anti-CD20 antibodies was measured by surface plasmon resonance (Biacore). For PMN activation and phagocytosis, we have used either purified PMN or analyzed PMN function in unmanipulated whole blood assays from normal donors or CLL patients. PMN activation was measured as CD11b upregulation and CD62L downmodulation by flow cytometry. Phagocytosis by PMN of chronic lymphocytic leukemia (CLL) cells was measured by triple fluorescence (PKH26, CD15-FITC and CD19-APC) and flow cytometry. Results Obinutuzumab or glycoengineered defucosylated rituximab (called G2) bound CD16B with about 7 fold higher affinity, compared to non-glycoengineered wild type parental antibodies. This was true either using surface plasmon resonance or measuring antibody binding to live PMN. Furthermore obinutuzumab activated PMN, either purified or in whole blood, more efficiently than RTX. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on PMN and in release of TNFα, IL-6 and IL-8. Activation was not accompanied by generation of reactive oxygen species or ADCC, but led to phagocytosis of anti-CD20 antibody opsonized CLL targets by purified PMN. Indeed up to 50% phagocytic PMN could be observed in presence of obinutuzumab or G2 antibodies after 6-24 hours incubation of purified PMN with CLL targets. Significant phagocytosis (15%) was also observed in whole blood, but only in presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using blocking F(ab) and F(ab’)2 fragments specific for CD16B and CD32A, that both these receptors are involved in PMN activation, phagocytosis and cell death induced by glycoengineered anti-CD20 antibodies. The possible effect of NA1 and NA2 polymorphisms of CD16B on obinutuzumab binding and phagocytosis is under further investigation. Conclusions We conclude that phagocytosis by PMN is an additional mechanism of action of obinutuzumab, mediated through its higher binding affinity for CD16B compared to RTX. Phagocytosis takes place in whole blood and is followed by PMN death. This effect may in part explain the neutropenia observed after treatment of B-CLL patients with GA101. Disclosures: Golay: Roche Glycart AG: Research Funding. Ferrara Koller:Roche Glycart AG: Employment. Rambaldi:Roche Italia: Consultancy, Honoraria. Klein:Roche Glycart AG: Employment. Introna:Roche Glycart AG: Research Funding.

We explored the molecular mechanisms involved in the establishement of CMA-03/06, an IL-6-independent variant of the multiple myeloma cell line CMA-03 previously generated in our Institution. CMA-03/06 cells grow in the absence of IL-6 with a doubling time comparable with that of CMA-03 cells; neither the addition of IL6 (IL-6) to the culture medium nor co-culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL-6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL-6 independence of CMA-03/06 cells is not apparently due to the development of an autocrine IL-6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA-03/06 cells showed an activated pattern of the NF-κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA-03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA-03/06 cells display a higher sensibility to NF-κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL-6 independence of CMA-03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell-line CMA-03 and its variant CMA-03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL-6-independent growth of myeloma cells.