Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality. We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness. Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14–17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use. Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach’s scalability and clinical potential.

Non-viral engineering can ease CAR-T cell production and reduce regulatory and cost requirements. We utilized Sleeping Beauty transposon to engineer donor-derived anti-CD19.CD28.OX40.CD3zeta T cells differentiated in cytokine-induced killer (CARCIK-CD19) for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (alloHSCT). We report the results of CARCIK-CD19 observed in 36 patients (4 children and 32 adults) treated according to the final recommended dose. Cytokine release syndrome of grade 2 or lower occurred in 15 patients, ICANS grade 2 in 1 patient, and late-onset peripheral neurotoxicity of grade 3 in 2 patients. GVHD never occurred after treatment with allogeneic CARCIK-CD19. Complete remission was achieved by 30 out of 36 patients (83.3%), with MRD negativity in 89% of responders. With a median follow-up of 2.2 years, the 1-year overall survival was 57.0%, and event-free survival was 32.0%. The median duration of response at 1 year was 38.6%. CAR-T cells expanded rapidly after infusion and remained detectable for over 2 years. Integration site analysis after infusion showed a high clonal diversity. These data demonstrated that SB-engineered CAR-T cells are safe and induce durable remission in heavily pretreated patients with BCP-ALL relapsed after alloHSCT. Trial registration: The phase 1/2 and phase II trials are registered at www.clinicaltrials.gov as NCT#03389035 and NCT#05252403.

Commercial CAR T cells typically employ autologous T cells, which can be functionally unfit due to patient age, prior treatments, and tolerance within the tumor microenvironment. The autologous approach has practical drawbacks too, including manufacturing failures, time-consuming production, and high costs, prompting the need for more feasible and efficient protocols. Efforts are now focused on developing allogeneic CAR T cell therapies, though challenges like Graft-Versus-Host Disease (GVHD) and limited persistence remain. Our work has proven feasibility and safety of using Peripheral Blood (PB) healthy donor cells to generate CARCIK-CD19 cells1. Cord blood (CB) is a standard hematopoietic stem cell source and offers immunological advantages such as reduced risk of GVHD2 and enhanced graft versus leukemia particularly in patients with pre-transplant residual disease. CB T cells exhibit greater proliferation compared to adult PB T cells, especially when stimulated with cytokines like IL-7 and IL-152. Cytokine-induced killer (CIK) cells, which are CD3+CD56+-enriched T cells, are easily expandable in vitro from PBMCs and are associated with minimal GVHD. CIK cells, similar to natural killer cells, possess non-MHC-restricted cytolytic activity and have proven to be safe and effective against various solid and hematologic malignancies, representing an alternative effector T cell source for adoptive immunotherapy3. We demonstrated that functional CIK cells can be derived from both fresh and cryopreserved CB units4,5. The CB rapid availability and the low risk of GvHD represent appealing features for the generation of banked third-party “off the shelf” CB-derived CARCIK cells. Fresh or thawed CB mononuclear cells were successfully modified with a third generation anti-CD19.CAR using a non-viral Sleeping Beauty (SB) transposon gene transfer platform optimized by our group, reaching up to 50% of CAR expression. Compared to the protocol for PB-derived CARCIK cells, we added a purification step to remove erythroblasts and used IL-7 and IL-15 instead of IL-2. These two variables significantly increased the cell yields of CARCIK cells obtained from frozen CB bags (from 5x108 to 1.6x109 total cells/CB bag subunit). Interestingly, we observed that CB-derived CARCIK cells are more metabolically fit. Using the NanoString CAR T Characterization Panel, we found differential gene expression profiles between PB and CB-derived CARCIK-CD19 cells. CB-derived cells exhibited a lower glycolysis score, validated by Seahorse analysis, and enhanced patterns of persistence, chemokine signaling, and T-cell migration. The in vitro functional profile of CB-derived CARCIK-CD19 was comparable to PB-derived ones. We then conducted three large-scale good manufacturing Practices (GMP)-grade validation runs using frozen CB bags. The total cell yields and CAR expression were 1.83 x 1010, 2 x 1010 and 2.5 x109 and 62.3%, 36.42% and 10.0% for the three respective runs. GMP-grade runs of both CB- and PB- derived CARCIKCD19 cells were functionally validated in vivo using a DAUDI xenograft NSG model, where both cell products prolonged the survival of treated mice and controlled disease progression. We are currently planning to incorporate these findings in our upcoming clinical studies with CARCIK cells. In conclusion, we have successfully demonstrated the feasibility of deriving functional CARCIK-CD19 cells from the cord blood (CB) source. Metabolic and transcriptomic analyses revealed that CB-CARCIK cells exhibit a lower glycolytic score and a higher memory score compared to those derived from peripheral blood (PB), indicating advantageous CAR T cell characteristics. Furthermore, we established the scalability of a GMP-grade manufacturing process for deriving CARCIK cells from CB, enabling the production of readily available, banked, third-party CARCIK cells for treating hematological malignancies. References: 1 Magnani CF, et al. J Clin Invest 2020; 130: 6021-6033. 2 Borrill R, et al. Front Pediatr 2023; 11: 1232281. 3 Schmeel LC, et al. J Cancer Res Clin Oncol 2015; 141: 839-49. 4 Introna M, et al. Bone Marrow Transplant 2006; 38. doi:10.1038/sj.bmt.1705503. 5 Introna M, et al. Biology of Blood and Marrow Transplantation 2010. doi:10.1016/j.bbmt.2010.05.015.

Background Traumatic brain injury (TBI) is a significant cause of death and disability, with no effective neuroprotective drugs currently available for its treatment. Mesenchymal stromal cell (MSC)-based therapy shows promise as MSCs release various soluble factors that can enhance the injury microenvironment through processes, such as immunomodulation, neuroprotection, and brain repair. Preclinical studies across different TBI models and severities have demonstrated that MSCs can improve functional and structural outcomes. Moreover, clinical evidence supports the safety of third-party donor bank-stored MSCs in adult subjects. Building on this preclinical and clinical data, we present the protocol for an academic, investigator-initiated, multicenter, double-blind, randomised, placebo-controlled, adaptive phase II dose-finding study aiming to evaluate the safety and efficacy of intravenous administration of allogeneic bone marrow-derived MSCs to severe TBI patients within 48 h of injury. Methods/design The study will be conducted in two steps. Step 1 will enrol 42 patients, randomised in a 1:1:1 ratio to receive 80 million MSCs, 160 million MSCs or a placebo to establish safety and identify the most promising dose. Step 2 will enrol an additional 36 patients, randomised in a 1:1 ratio to receive the selected dose of MSCs or placebo. The activity of MSCs will be assessed by quantifying the plasmatic levels of neurofilament light (NfL) at 14 days as a biomarker of neuronal damage. It could be a significant breakthrough if the study demonstrates the safety and efficacy of MSC-based therapy for severe TBI patients. The results of this trial could inform the design of a phase III clinical trial aimed at establishing the efficacy of the first neurorestorative therapy for TBI. Discussion Overall, the MATRIx trial is a critical step towards developing an effective treatment for TBI, which could significantly improve the lives of millions worldwide affected by this debilitating condition. Trial Registration EudraCT: 2022-000680-49.

Innovative pro-regenerative treatment strategies for progressive multiple sclerosis (PMS), combining neuroprotection and immunomodulation, represent an unmet need. Neural precursor cells (NPCs) transplanted in animal models of multiple sclerosis have shown preclinical efficacy by promoting neuroprotection and remyelination by releasing molecules sustaining trophic support and neural plasticity. Here we present the results of STEMS, a prospective, therapeutic exploratory, non-randomized, open-label, single-dose-finding phase 1 clinical trial ( NCT03269071 , EudraCT 2016-002020-86), performed at San Raffaele Hospital in Milan, Italy, evaluating the feasibility, safety and tolerability of intrathecally transplanted human fetal NPCs ( hf NPCs) in 12 patients with PMS (with evidence of disease progression, Expanded Disability Status Scale ≥6.5, age 18–55 years, disease duration 2–20 years, without any alternative approved therapy). The safety primary outcome was reached, with no severe adverse reactions related to hf NPCs at 2-year follow-up, clearly demonstrating that hf NPC therapy in PMS is feasible, safe and tolerable. Exploratory secondary analyses showed a lower rate of brain atrophy in patients receiving the highest dosage of hf NPCs and increased cerebrospinal fluid levels of anti-inflammatory and neuroprotective molecules. Although preliminary, these results support the rationale and value of future clinical studies with the highest dose of hf NPCs in a larger cohort of patients.

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 <-150 degrees C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. Conclusions: Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion. (c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Background: CD19-targeted Chimeric Antigen Receptor (CAR) T cell therapy has shown unprecedented results in patients with relapsed/refractory acute lymphoblastic leukemia (R/R ALL) where no other curative options are available. Despite a complete remission (CR) rate of 80%, more than half of the adult patients eventually relapse. Which patients are likely to benefit from consolidative treatment remains to be addressed. We conducted an academic, multi-center, phase I/II dose-escalation trial in ALL patients relapsed after allogeneic hematopoietic cell transplantation (HCT). Patients received donor-derived, CD19-targeted CAR cytokine induced killer (CARCIK-CD19) cells engineered with the non-viral Sleeping Beauty transposon vector (Magnani et al. JCI, 2020). Objective: The aims of this study were to evaluate the impact on clinical outcome of proliferation, differentiation, and expansion of infused CARCIK-CD19 cells. Methods: Data were prospectively collected from consecutive patients enrolled in the FT01CARCIK, Phase I/IIb study (NCT03389035), and a compassionate use study (FT02-CARCIKCD19). Patients underwent fludarabine and cyclophosphamide-based lymphodepletion, before CARCIK-CD19 infusion. For this study, only patients receiving cell doses previously shown to be well tolerated and effective (7.5x106/Kg [DL3] and 15 x106/Kg [DL4]) were included. CAR+ T cells were counted in the peripheral blood (PB) at predefined time points, using flow cytometry (FC). We assessed the distribution of T cell maturation subsets in infused CAR+ T cells, identified as: naïve (CD45RA+CD62L+), central memory (CM, CD45RA-CD62L+), effector memory (EM, CD45RA-CD62L-) T cells, and terminal differentiated T cells (TEMRA, CD45RA+CD62L-). Minimal residual disease (MRD) monitoring was performed using both FC and quantitative PCR (qPCR), in PB and bone marrow (BM). CR was defined as absence of leukemia in the BM at day 28. CARCIK-CD19 peak was defined as the maximum amount of CAR+ T cells/μL measured in PB. CAR+ T cell persistence was defined as any CAR+ T cell detected after 90 days, expressed as cells/μL. Duration of response (DOR) was defined as persistence of response after achieving CR at day 28. Results: This analysis included 20 patients treated with CARCIK-CD19 cells from October 2018 to October 2021, 15 of whom were enrolled in the Phase I/IIb study and 5 patients in the subsequent compassionate use study. CR rate at day 28 was 76.2% (95% CI= 52.8-91.8%), of which 81.3% were MRD negative. The median OS was 12 months. The OS at 6 months was 71.4%. Circulating CAR+ T cells peaked at day 10, with a median of 48.5/μL (range, 0.6-718), whereas the median value at day 28 was 2.6/μL (range, 0-33.2). CAR+ T cells were still detectable at month 12 with a median of 0.8/μL (range 0-2.2) (Figure 1). As shown in Figure 1, the majority of CAR+ T cells were CD8+ T cells. At day 7 the majority of CAR+ T cells were CM with a median of 31.7% (range, 0.7-90) and EM T cells with a median of 25% (range 7-96.5), but we still observed a high percentage of Naïve CAR+ T cells, with a median of 9.9% (range, 0-47.9) and TEMRA, median 4.5% (range 0-32.9). Instead, on day 28 the majority of CAR+ T cells were EM, with a median of 33.7% (range, 0-96.8), followed by CM T cells, median 22.1% (range, 0-77.8), Naïve, median 10.2% (range, 0-66.66), and TEMRA, median 10% (range, 0-49.9). A negative MRD status in the BM at day 28 assessed by FC positively correlated with OS (p=0.004). In addition, the persistence of CAR+ T cells after day 90 correlated with DOR (p= 0.0045, Figure 2). Conclusions: In this study we showed that CARCIK-CD19 cells efficiently expanded in vivo, reaching the peak at day 10, and persisting in some cases until 12 months after infusion. The majority of CAR+ T cells were CD8+ with a memory phenotype confirming the capability of these cells to persist long-term. CAR+ T cells persistence was associated with a longer duration of response and reduced risk of leukemia relapse. The achievement of a negative MRD status at day 28 was crucial for patient's survival, suggesting the need of early additional treatment in patients failing this end point. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 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.

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 CART 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 CART 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 CART 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. CART 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 CART cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.

Background: Immunotherapy using 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. Unmodified allogeneic Cytokine Induced Killer (CIK) cells (CD3+CD56+ T cells) have clearly demonstrated a high profile of safety in ALL patients. Aims: Here, we demonstrate the feasibility and reproducibility of a good manufacturing practices (GMP)-compliant culture of allogeneic CIK cells modified by non-viral Sleeping Beauty (SB) transposon to obtain CD19CAR T cells for the clinical application. Preliminary analysis of CARCIK-CD19 cellular kinetic in 6 patients are also reported. Methods: PBMCs were electro-transferred with the SB GMP-grade CD19.CAR/pTMNDU3 plasmid and pCMV-SB11 plasmid (kindly provided by L. Cooper, Houston). CIK cells were then generated according to the method enclosed in the filed patent EP20140192371. The manufacturing process were performed in a academic cell factory authorized by Agenzia Italiana del Farmaco (AIFA). CARCIK-CD19 were infused in pediatric and adult B-ALL patients relapsed post transplantation after standard lymphodepletion. Results: We manufactured ten batches by seeding a median of 103.16x106 allogeneic PBMCs derived from 50 ml of PB. After 20–28 days of culture (median 22) we harvested a median of 3.6x109 nucleated cells (range 1.40 – 15.75x109). At the end of expansion, cell viability was 97.24% (range 91.99%>98.96%), manufactured cells were mostly CD3+ lymphocytes (mean 98.73% ± SD 1.55%). Of these, 46.17% ± 17.92% were CAR+ and 43.89% ± 10.13% were CD56+, while median fold increase was 176.6 (37.0–1350) and had a median vector copy number (VCN) of 3.5 VCN/cells. In all the ten batches, CARCIK-CD19 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19+ REH target cell line (mean 80.68%, range 61.89%>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 using Sonication Linker Mediated (SLiM)-PCR. All the batches were released after about 10 days after the end of production. The quality requirements for batch release were met in all 10 productions. CARCIK-CD19 achieved robust expansion in the majority of the patients as defined by detectable CAR T-cell detection by VCN (range 4645–343403 transgene copies/ug) and flow (range 0.5–30%) in the blood. The median time to peak engraftment in peripheral blood was 14 days. The magnitude of expansion in peripheral blood correlate with the disease burden at the time of product infusion in the marrow. Summary/Conclusion: Overall, these results demonstrate that clinical-grade SB transduction of allogeneic CIK cells with CD19 CAR is feasible and allows efficient expansion of CARCIK-CD19 cells starting from easily available small amounts of PB, with important implications for non-viral technology. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing and demonstrates expansion of CARCIK-CD19 post infusion (NCT03389035).

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.

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).

Cell therapy with bone marrow (BM)-derived progenitors has emerged as a promising therapeutic for refractory angina (RA) patients. In the present study, we evaluated the safety and preliminary efficacy of transcatheter delivery of autologous BM-derived advanced therapy medicinal product CD133+ cells (ATMP-CD133) in RA patients, correlating perfusion outcome with cell function.

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.

CD19-specific CART cells demonstrated unexpected positive results, achieving complete remission and durable response in relapsed and refractory patients affected by B-lineage neoplasms. We recently established a platform for non-viral gene manipulation of Cytokine-Induced Killer (CIK) cells, an effector T cell population characterized by enrichment in highly cytotoxic CD3+CD56+ cells and reduced risk of GvHD, in compliance with Good manufacturing practices (GMP).