Abstract: 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 & lt; 5% bone marrow (BM) blasts, circulating blasts & lt; 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.

Abstract: Background: Acute lymphoblastic leukemia (ALL) is a malignant disorder with a long-term remission of less than 50% of adult patients and of nearly 80% of children. Relapsed and refractory (r/r) adult and childhood B-ALL patients, have significant unmet medical needs. Adoptive transfer of patient-derived T cells engineered to express a chimeric antigen receptor (CAR) by viral vectors has achieved complete remission and durable response in highly refractory populations (June CH et al. Science 2018). In addition, unmodified Cytokine Induced Killer (CIK) cells (CD3+, CD56+ T cells) have clearly demonstrated a high profile of safety in ALL patients (Introna M et al. Biol Blood Marrow Transplant. 2017). Here, we demonstrate the feasibility and reproducibility of a GMP-compliant clinical-grade culture and gene-modification protocol of allogeneic CIK cells using the non-viral Sleeping Beauty (SB) transposon system (Singh H et al, Plos One 2013) to obtain CD19CAR expressing CIK cells (Magnani CF et al, Oncotarget 2016, Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017) starting from a limited amount of an easily available material such as peripheral blood (PB). Methods: Fifty mL of PB were centrifuged on Ficoll gradient to obtain mononuclear cells (PBMCs). PBMCs were then simultaneously electro-transferred with the SB GMP-grade DNA transfer CD19.CAR/pTMNDU3 plasmid (human 3rd generation anti-CD19CD28OX40z CAR under the pTMNDU3 promoter), and transposase pCMV-SB11 plasmid (kindly provided by L. Cooper, MDACC, Houston, TX, USA). CIK populations (Introna M et al, Haematologica 2007) were then generated according to the method enclosed in the filed patent EP20140192371 (Magnani CF et al, Oncotarget 2016). The manufacturing process and the quality control tests were performed in a good manufacturing practices (GMP) academic cell factory authorized by Agenzia Italiana del Farmaco (AIFA) in the context of an ongoing phase I clinical trial (NCT03389035) for children and adults with relapsed/refractory B-cell precursor ALL post hematopoietic stem cell transplantation (HSCT). Results: We manufactured nine batches by seeding a mean of 102.52x106 allogeneicPBMCs derived from 50 ml of peripheral blood (range 46.1 - 193.17x106). After 21-22 days of culture the mean harvesting was 5.0x109 nucleated cells (range 1.15 - 16.00x109) with a mean viability of 97.56% (min. 95.24% - max 99.43%). These cells were mostly CD3+ lymphocytes (mean 98.54%, min. 94.85% - max 99.68%) which had a median fold increase of 87.3. Expanded CD3+ cells expressed CD56+ and surface CAR at variable levels among the batches (mean 44.79% and 43.78%, respectively) and had a median vector copy number (VCN) of 2.56 VCN/cells, according to pre-clinical data (Magnani CF et al, Hum Gene Ther. 2018). In all the nine batches, CARCIK-CD19 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19+ REH target cell line (mean 82.96%, min. 61.89% - max 97.72%). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by integration site (IS) analysis performed by Sonication Linker Mediated (SLiM)-PCR and Illumina MiSeq sequencing. The GMP batches were released after about 10 days after the end of production. Quality control release specifications and results are reported in Table 1. Conclusions: Overall, these results demonstrate that clinical-grade SB transduction of allogeneic CIK cells with CD19 CAR is feasible and allows rapid and efficient expansion of highly potent CARCIK-CD19 cells starting from easily available small amounts of PB, with important implications for non-viral technology. In summary our data represent a solid ground for the future development of further SB-based platforms. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing (NCT03389035). Disclosures Gritti: Autolus: Consultancy. Rambaldi:Celgene: Consultancy; Omeros: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Pfizer: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy.

Abstract: Transplantation of human bone marrow mesenchymal stromal cells (hBM-MSC) promotes functional recovery after stroke in animal models, but the mechanisms underlying these effects remain incompletely understood. We tested the efficacy of Good Manufacturing Practices (GMP) compliant hBM-MSC, injected intravenously 3.5 hours after injury in mice subjected to transient middle cerebral artery occlusion (tMCAo). We addressed whether hBM-MSC are efficacious and if this efficacy is associated with cortical circuit reorganization using neuroanatomical analysis of GABAergic neurons (parvalbumin; PV-positive cells) and perineuronal nets (PNN), a specialized extracellular matrix structure which acts as an inhibitor of neural plasticity. tMCAo mice receiving hBM-MSC, showed early and lasting improvement of sensorimotor and cognitive functions compared to control tMCAo mice. Furthermore, 5 weeks post-tMCAo, hBM-MSC induced a significant rescue of ipsilateral cortical neurons; an increased proportion of PV-positive neurons in the perilesional cortex, suggesting GABAergic interneurons preservation; and a lower percentage of PV-positive cells surrounded by PNN, indicating an enhanced plastic potential of the perilesional cortex. These results show that hBM-MSC improve functional recovery and stimulate neuroprotection after stroke. Moreover, the downregulation of “plasticity brakes” such as PNN suggests that hBM-MSC treatment stimulates plasticity and formation of new connections in the perilesional cortex.

Abstract: Flow‐cytometric monitoring of minimal residual disease (MRD) in bone marrow (BM) during induction of pediatric patients with acute lymphoblastic leukemia (ALL) is widely used for outcome prognostication and treatment stratification. Utilizing peripheral blood (PB) instead of BM might be favorable, but data on its usefulness are scarce. Procedure We investigated 1303 PB samples (days 0, 8, 15, 33, and 52) and 285 BMs (day 15) from 288 pediatric ALL patients treated in trial AIEOP‐BFM ALL 2000. MRD was assessed by four‐color flow cytometry and evaluated as relative, absolute, and kinetic result. Results In B‐ALL only, PB measures from early time points correlated with relapse incidence (CIR). Best separation occurred at threshold 〈 1 blast/μL at day 8 (5‐year CIR 0.02 ± 0.02 vs 0.12 ± 0.03; P  = 0.044). Patients with highest relapse risk were not distinguishable, but PB‐MRD at days 33 and 52 correlated with prednisone response and postinduction BM‐MRD by PCR ( P   〈  0.001). Kinetic assessment did not convey any advantage. In multivariate analysis including day 15 BM‐MRD, PB‐MRD measures lost statistical power. Conclusions In summary, PB‐MRD in pediatric B‐ALL correlates with outcome and risk parameters, but its prognostic significance is not strong enough to substitute for BM assessment in AIEOP‐BFM trials. It might, however, be valuable in treatment environments not using multifaceted risk stratification with other MRD measures.

Abstract: Background Multiparametric flow cytometry (MFC) is critical in the diagnosis and management of pediatric acute lymphoblastic leukemia (ALL), through immunophenotyping (IP), and minimal residual disease (MFC MRD) analysis. The aberrant expression of myeloid markers in B- and T-lineage ALL is a well-known phenomenon. It may be the result of an adaptive mechanism by lineage switch (SW), defined as any variation of blast IP over time. CD371 is a transmembrane glycoprotein usually expressed on normal myeloid cells and most of the myeloid blasts. Aberrant expression of CD371 was observed in DUX4-rearranged B cell precursor ALL (BCP ALL). Aims To investigate the clinical and biological features of CD371 positive (CD371+) pediatric BCP ALL, pointing out its potential implication in MFC MRD monitoring on Day 15. Materials and Methods From June 2014 to January 2017, 862 children with newly diagnosed t(9;22)(q34.1;11.2);BCR-ABL1 negative BCP ALL, were consecutively enrolled in the AIEOP BFM ALL 2009 study by AIEOP centers. Peripheral blood (PB) and bone marrow (BM) samples (SMPs) were processed and analyzed in the Laboratory of Diagnosis and Research of Pediatric Hematology-Oncology, University of Padua, Italy, according to standardized operating protocols designed by the AIEOP BFM Flow Network. At diagnosis, 9 combinations of 8 monoclonal antibodies (MoAbs) were used for IP. MFC MRD (Day 8 on PB, Day 15, 33, and 78 on BM) was performed with 2 combinations of 8 MoAbs from June 2014 to May 2016. Later, a dry 10 colors preformulated tube was adopted for MFC MRD monitoring. Results At diagnosis, CD371 expression was assessed in 823 of 862 (95.5%) SMPs by as many patients (pts; age: 1-17 years; male/female: 446/377). Of those, CD371 was positive in 75 of 823 SMPs (9.1%). CD371 positivity was associated with older age, euploidy, a more immature immunophenotype (B-I as per EGIL classification), and the aberrant expression of CD2 antigen, as well as at least one myeloid marker (Table). CD371+ SMPs showed a stronger expression of CD34, CD45, and CD58 antigens than CD371 negative SMPs (Table). We performed MFC MRD analysis on 207 SMPs of CD371+ BCP ALL (42 on Day 8, 72 on Day 15, 40 on Day 33, and 53 on Day 78). During the first 15 days of Induction therapy, a monocytoid population appeared in 76 SMPs [26 of 42 (61.9%) on Day 8 and 50 of 72 (69.4%) on Day 15] . It was characterized by a strong expression of CD34, CD58 and CD45, reduced expression of CD19, and high SSC. We interpreted that phenomenon as an SW to the myelomonocytic lineage, as previously described in a subtype of BCP ALL expressing CD2 at diagnosis. Myelomonocytic SW displayed 2 different patterns: (a) a single population of blasts with heterogeneous expression of CD19 (strong to dim/negative); (b) 2 distinct populations: the first one with the IP of diagnosis, the second one showing a downregulation of CD19 and CD34, an intensification of CD45, and an increase of SSC (Figure). At the same time points, a clear monocytoid population was visible on May-Grunwald-Giemsa stained smears. The comparison between MFC MRD and PCR MRD data showed a higher concordance when both the populations were included in the final amount of blasts on Day 15 (concordance rate: 89% vs. 82%). SW was transient and disappeared after Day 15, even though chemotherapy was always carried on as per therapeutic ALL protocol. CD371 antigen was an accurate marker of SW in our cohort [sensitivity = 0.93 (95% CI ± 0.06), specificity = 0.98 (95% CI ± 0.005), accuracy = 0.98]. Finally, CD371 positivity was associated with a worse response to Induction therapy, showing a higher proportion of pts enrolled in the high therapeutic risk group of the trial, most frequently due to a slow early response according to PCR MRD on Day 33 and 78 (Table). Conclusions We described a new subtype of pediatric BCP ALL, characterized by the aberrant expression of CD371 and a potential myelomonocytic SW during the first phase of Induction Therapy. Accurate identification of the lineage SW is mandatory to properly assess MFC MRD on Day 15 in these pts, avoiding an underestimation of blast cells. This is particularly important, considering that CD371+ BCP ALL was associated with a poor response to Induction therapy. Even in presence of a prevalent monocytoid population, chemotherapy should be carried on according to a therapeutic protocol for ALL. CD371 antigen should be part of IP diagnosis panel for ALL. A multicenter study of AIEOP BFM Flow Network centers is ongoing. Disclosures Brüggemann: Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy.

Abstract: Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP‐BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi‐color FCM: marker panel requirements, immunological blast gating, in‐sample controls, tri‐partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish “simple” from bilineal/“complex” mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter‐laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society

Abstract: Whole-exome sequencing reveals the presence of recurrent somatic mutations of ETNK1 in patients with atypical chronic myeloid leukemia. ETNK1 mutations impair the catalytic activity of the enzyme, causing a decrease in the intracellular levels of phosphoethanolamine.