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.

Abstract: 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 & 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 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).

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: Introduction Cytokine Induced Killer (CIK) cells are memory T lymphocytes which have acquired CD56 expression and Natural Killer (NK) like unrestricted cytotoxicity, following in vitro activation by anti CD3 OKT3 and IFNg and subsequent expansion with IL-2. CIK cells have demonstrated in vitro and in vivo anti tumor activity, direct intratumor homing following iv. administration and, more importantly, a very reduced Graft Versus Host (GVH) activity, in several experimental allogeneic models. Indeed we and others have demonstrated very limited GVH activity in preliminary phase I studies, with donor derived (matched) CIK cells in patients with different hematological neoplasms, previously treated by allogeneic Hematopoietic Stem Cells (HSC) transplantations and subsequently relapsed of diseases 1. Methods To better delineate the toxicity profile, as well as the potential anti tumor efficacy, of donor derived CIK cells, we prospectively studied 48 patients relapsed after allogeneic stem cell transplantation performed using either a matched related (N=28) or unrelated donor (n= 20, including 1 haplo). This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, approved by the Agenzia Italiana del Farmaco and (AIFA). The trial was registered as (EUDRACT n 2008-003185-26, ClinicalTrial.gov: NCT01186809) Results In this interim analysis, forty-eight patients (including childrens and adults) have been so far enrolled into this study protocol. The median age was 48 (range 6-67) and a diagnosis of ALL (n=9, 20%), AML (n=29, 60%), MM (n=5, 8%), HD (n=3, 6%), MPN (n=2, 4%), NHL (n=1, 2%). Reasons for being enrolled into study was a hematologic relapse in 36 (75%) or a molecular relapse in 12 (25%). The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with growing numbers of CIK cells, in groups of three patients per dose level. Since DLT was never observed (acute GVHD of grade III or more) the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to subsequent consecutive 36 patients. 4 patients died for disease progression and 1 patient developed aGVHD (grade I, skin only) during the DLI treatment and could not proceed to the planned subsequent CIK administration. Of the 43 patients who eventually received at least one infusion of CIK cells, 15 patients did not complete the program, 9 for disease progression and death, 3 for insurgence of grade II aGVHD (skin only in 2 cases, skin and gut in 1 case), 1 for hemolytic anemia, 1 for insufficient cell supply and 1 for medical decision. Overall, 28 patients received the complete cell therapy planned (58%). Overall, of the 48 patients enrolled, 5 (10%) suffered from aGVHD (1 grade I, 3 grade II, 1 grade III). During follow up, chronic GVHD was observed in 7 patients (14 %) (3 mild and 4 moderate). As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 13 (27%) patients (4 during the DLI), before the clinical response could be evaluated. A CR was observed in 9 (19 %) and a PR in 7 (14%) for an overall response rate of 16 (33%). No response was observed in 19 (39%). At 2 and 4 years, the event free survival of the 48 patients is 22% and 18%, while the overall survival is of 37% and 34%, respectively. For the small group of patients who achieved a complete response, the disease free survival is of 64% at 2 years and 51% at 4 years. By univariate analysis, survival was significantly associated to the type of relapse (molecular) (p 0.0081) since at 2 and 4 years it was and 24% and 27% vs. 71% and 71 % for patients enrolled for a hematologic or a molecular relapses, respectively. By multivariate analysis, the type of relapse remained the only significant predictor of survival (0.0160 p value). Conclusion This study shows the feasibility of CIK preparation and administration as well as the relatively low toxicity of the program (10% aGVHD grades I-III) in spite of the fact that 20 patients received cells from matched unrelated donors. Finally, the study offers the suggestion that CIK cells may be efficacious to treat post-transplant relapse. 1 Introna M. et al, Haematologica, 2007, 92, 7, 948. Figure 1. Figure 1. Disclosures Introna: roche: Research Funding. Rambaldi:Roche: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Celgene: Research Funding; Pierre Fabre: Honoraria.