Median Vector Copy Number Research Articles

Phase 1 Study of CART-Ddbcma for the Treatment of Patients with Relapsed and/or Refractory Multiple Myeloma: Results from at Least 1-Year Follow-up in All Patients

Introduction: CART-ddBCMA, an autologous anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapy with a unique, synthetic binding domain, is being studied in a first-in-human clinical trial in patients (pts.) with relapsed &/or refractory multiple myeloma (RRMM). One-year or more follow-up clinical data from all patients are presented in this report. Methods: Details of study design have been previously reported (Frigault et al Blood Adv 2023). Briefly, pts with RRMM who have received ≥3 prior lines of therapy were enrolled & received a single infusion of CART-ddBCMA following lymphodepletion chemotherapy (fludarabine: 30 mg/m 2/d & cyclophosphamide: 300 mg/m 2/d daily for 3 days). Two dose levels (DL1 & DL2 respectively) of 100 & 300 (±20%) ×10 6 CAR+ cells were evaluated. The primary endpoints included incidence of adverse events (AEs) & dose-limiting toxicities (DLTs). Additional endpoints included quality & duration of clinical response assessed according to the IMWG Uniform Response Criteria for MM, evaluation of minimal residual disease (MRD), progression-free (PFS) & overall survival (OS). Correlative analyses related to CART-ddBCMA expansion, characterization, patient & disease-related features were also evaluated. Results: As of June 2, 2023, 40 pts; median age 66 years (range: 44-76) were enrolled; 38 received CART-ddBCMA (32, DL1; 6, DL2) & 38 were evaluable for initial safety & clinical response. Two pts who were not dosed had cell product manufactured but were not eligible for cell infusion due to medical complications. Pts had a median of 4 (range: 3-16) prior lines of therapy. All infused pts (100%) were triple-refractory, & 26 (68%) were penta-refractory; 34 pts (89%) were refractory to last-line of treatment; 9 pts (24%) had high tumor burden with ≥60% bone marrow plasma cells; 13 (34%) pts had extramedullary disease; & 11 (29%) pts had high-risk cytogenetics (Del 17p, t(14;16), t(4;14)) at baseline. Median follow-up after CART-ddBCMA infusion was 22 months (range: 9-40 months). CAR+ cells comprised a median 70% (range: 48-87%) of total CD3+ T cells; median vector copy number was 2.2 copies/cell (range: 1.1-3.5); median cell viability was 98% (range: 90-100%), & median cell manufacturing yield was 1174 ×10 6 CAR + cells (range: 470-1626 ×10 6). CART-ddBCMA product characteristics were consistent with the specifications in all the lots, & there were no manufacturing failures. CRS occurred in 36/38 (95%) pts but only 1 pt in DL2 had grade (Gr) 3 CRS & all other cases were Gr≤2. ICANS occurred in 7 pts (5, Gr≤2; 2, Gr3), with 1 Gr3 case in each DL. All cases of CRS & ICANS resolved without further sequalae with management. No cases of off-tumor cell mediated toxicity, delayed neurotoxicity events (i.e., occurring after day 28), or Parkinsonian-like symptoms were observed. All 38 evaluable pts demonstrated investigator-assessed clinical response per 2016 IMWG criteria (ORR, 100%) with 22 sCR, 7 CR (≥CR rate, 76%), 6 VGPR (≥VGPR rate, 92%), & 3 PR. Responses deepened over time & conversion to CR/sCR was observed with longer follow-up (as late as month 12). Of those evaluable for MRD testing to date (n=29), 25 (86%) were MRD-neg at 10 -5. Median duration of response, PFS, & OS were not reached at the time of data-cut because 25 of 38 evaluable pts (66%) had ongoing response. The Kaplan-Meier estimated PFS rates for 6, 12 & 18 months were 92%, 74%, & 67% respectively. Durable responses were also observed in patients with high-risk features (EMD, BMPC ≥ 60%, or B2M ≥ 5.5 at baseline) & high-risk cytogenetics. PFS rates at 6-, 12-, & 18-months are shown in Table 1. Based on the results from the study, a dose of 115 ± 10 ×10 6 cells, consistent with DL1, was recommended for the phase 2 study. Conclusions: Adverse events with CART-ddBCMA, including CRS & ICANS, were manageable & no off-tumor tissue-targeted toxicity, delayed neurotoxicity, or Parkinsonian-like events were observed in the entire cohort at the time of data-cut. Ongoing efficacy results are encouraging, with 100% ORR, including 35 (92%) response of VGPR or better & 29 (76%) with CR/sCR. More importantly, clinical responses were durable with an overall estimated 18-mo PFS rate of 67% with comparable clinical responses seen in ‘high-risk’ patients known to have poor prognosis. Updated data with additional follow-up based on later data-cut will be presented.

Lovo-cel (bb1111) Gene Therapy for Sickle Cell Disease: Updated Clinical Results and Investigations into Two Cases of Anemia from Group C of the Phase 1/2 HGB-206 Study

Background: Lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) uses autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbAT87Q. The ongoing phase 1/2 HGB-206 study (NCT02140554) is the largest clinical trial of GT in SCD to date. We report updated efficacy and safety results for Group C and describe the results of additional investigations in two patients (pts) in Group C with persistent anemia. Methods: Pts (≥12-≤50 yrs of age) with SCD and recurrent severe vaso-occlusive episodes (VOEs) underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. Lab evaluations, SCD-related outcomes, and safety were assessed. Data are median (min-max) unless otherwise stated; all timepoints are from lovo-cel infusion. Results: As of July 2021, 35 Group C pts had received a lovo-cel infusion, with a median follow-up of 20.9 months (min-max: 8.5-28.5). Neutrophil and platelet engraftment were achieved on Days 20 (12-35) and 36 (18-136), respectively. The median vector copy number in peripheral blood remained stable in all pts at ≥1 c/dg from 6 months after infusion until the last study visit, and resulted in median HbAT87Q levels of ≥5 g/dL during this period. The median total Hb level increased without transfusions from 8.5 g/dL at baseline to ≥11 g/dL from 6 months through last visit; HbAT87Q contributed at least 40% of total Hb (Fig 1A). Of the 29 pts who could be evaluated, 28 had a complete resolution of severe VOEs after lovo-cel infusion in the 2-yr parent study as of last follow-up, as compared with 3.5 events per yr (1.0-13.5) in the 2 yrs before enrollment (Fig. 1B); no severe VOEs have been reported during the long-term follow up study. Key hemolysis markers approached normal levels. Overall, 15 pts (43%) had ≥1 serious adverse event after lovo-cel infusion; the most frequently reported were abdominal pain, drug withdrawal syndrome (opiate), nausea, vomiting, and dehydration (6% each). No cases of veno-occlusive liver disease, graft failure, replication-competent lentivirus, vector-mediated insertional oncogenesis, or hematologic malignancies were observed. The lovo-cel treatment regimen generally reflected the known side effects of HSPC collection and busulfan conditioning regimen, and/or adverse events commonly seen in the population being evaluated. Further investigations were conducted as of March 2022 in two pts who presented with similar clinical and pathological findings that initially raised suspicion of myelodysplastic syndrome (MDS). Presentation features included neutropenia in one patient; both patients had persistent anemia despite adequate HbAT87Q production, bone marrow morphology showing erythroid specific dysplasia (binucleated or trinucleated cells) consistent with dyserythropoiesis as seen in other hemoglobinopathies, and low level of trisomy 8 (≤7.7%) only observed by fluorescence in situ hybridization analysis. There are no clinical symptoms suggestive of hematologic malignancies, no driver mutations, no clonal process (vector-related or otherwise) as shown by Integration Site Analysis and Next-Generation Sequencing (both subjects had >29,000 Unique Integration Sites with no integration having a relative frequency above 0.425%), and no chromosomal abnormalities on karyotyping among up to 205 metaphases. Both pts have had anemia and one maintained total Hb of 9.8 g/dL at Month 18 without transfusions, and the other required chronic transfusions to maintain 10.3 g/dL at Month 18 with otherwise normal complete blood counts. Both pts experienced a reduction or resolution of severe VOEs after lovo-cel infusion. Of note, these are the only pts in the study to have two α-globin deletions (−α3.7/−α3.7). We propose that α-globin trait may contribute to anemia and dyserythropoiesis after lovo-cel infusion. Summary: In Group C of the HGB-206 study, one-time treatment with lovo-cel resulted in sustained production of HbAT87Qand near-complete resolution of severe VOEs. Investigations to date in both patients with anemia have not confirmed MDS; thus, currently no hematologic malignancies have occurred in Group C. Ongoing long-term follow-up should be continued for all pts receiving GT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lentiviral Gene Therapy with Low Dose Conditioning for X-Linked SCID Results in Complete Immune Reconstitution and No Evidence of Clonal Expansion

Introduction: Gene therapy (GT) for X-linked severe combined immunodeficiency (SCID-X1) using a gamma (γ) retroviral vector resulted in robust T cell immune reconstitution but also insertional oncogenesis. A multi-institutional trial using a self-inactivating (SIN) γ-retroviral vector resulted in equivalent T cell immune reconstitution but no insertional oncogenesis to date (Hacein-Bey-Abina et al, NEJM 2014). In both trials, conditioning was only rarely used and therefore gene marking in B cells and neutrophils was negligible and reconstitution of NK cells was limited. To improve both the immune reconstitution and safety of GT for SCID-X1, we developed a lentiviral vector (LV) in which a codon optimized IL2RG transgene is driven by the elongation factor 1 alpha short promoter (EFS) and introduced low dose busulfan conditioning to enhance hematopoietic and multilineage expression of IL2RG. An international, multicenter, Phase 1/2 clinical trial (NCT03311503, NCT03601286) is currently underway to evaluate the safety and efficacy of LV mediated gene transfer using this vector. Methods: A total of 14 patients have been enrolled. Two patients were withdrawn due to poor stem cell mobilization and collection and manufacturing are in progress for 3 patients. Nine patients have been infused, 8 receiving mobilized peripheral blood stem cells and 1 receiving bone marrow at an average age of 5.3 months (range 3.2-7.1). Hematopoietic stem cells (CD34+ cells) were transduced with the LV vector (using transduction enhancers in 7 products) and drug products were cryopreserved. Median cell dose was 9.06 x 10e6 CD34+ cells/kg (4.55-17.87) and median vector copy number (VCN) was 0.86 copies/diploid genome(dg) (0.67-2.79). Drug products were thawed and infused after 2 days of busulfan conditioning targeted to an AUC of 30 mg*h/L. All infants tolerated busulfan exposure without any significant toxicity. Results: As of July 2022, all treated patients are alive with robust reconstitution of gene marked T cells with median follow up of 2.2 years (0.2-4.3). T cell reconstitution was rapid with median CD3+ T cell count 552 cells/uL (132-1160) at 3 months. Seven patients evaluable at 1 year had CD3+ (2377-4900 cells/uL) and CD4+ T cell counts (1507-3080 cells/uL) normal for age at 1-year post-GT (Figure 1). Gene marking in T cells was robust averaging 1.55-5.66 copies/dg. Evidence of thymic function includes rising naïve CD4+ T cells and maintenance of a normal CD4/CD8 ratio. Multilineage engraftment of gene modified cells is evident in B cells, neutrophils, and NK cells at last follow-up with stable VCN over time (ranges 0.43-2.05, 0.24-2.11, 1.98-3.61 respectively). In contrast to limited NK cell reconstitution seen in the absence of conditioning in prior trials, NK cell counts have been sustained in 6 of 7 patients with greater than 12 months of follow-up achieving values within the normal range for age (Figure 1). Six of these 7 patients have been able to stop immunoglobulin replacement, 4 of 4 who have completed a primary vaccine series show response, with the results of 2 further patients awaited. Insertion site analysis shows uniformly polyclonal repertoire with no evidence of clonal expansion. Conclusion: In summary, gene therapy using a lentiviral vector and low dose busulfan conditioning resulted in robust and complete immune reconstitution correcting T, NK, and B cell defects characteristic of SCID-X1 with 100% survival and no gene therapy related adverse events. Figure 1 Immune reconstitution in 7 patients treated with >1 year follow-up A) CD3+ T cells. B) CD4+ T cells. C) NK cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interim Results from a Phase 1/2 Clinical Study of Lentiglobin Gene Therapy for Severe Sickle Cell Disease

Interim Results from a Phase 1/2 Clinical Study of Lentiglobin Gene Therapy for Severe Sickle Cell Disease

Interim Results from a Phase I/II Clinical Gene Therapy Study for Newly Diagnosed Infants with X-Linked Severe Combined Immunodeficiency Using a Safety-Modified Lentiviral Vector and Targeted Reduced Exposure to Busulfan

▪Early trials of gene therapy for X-linked Severe Combined Immunodeficiency (XSCID) restored T cell immunity in most cases, but did not correct B cell function and carried a high risk of iatrogenic leukemia. The subsequent development of self-inactivating γ-retroviral vectors has enhanced safety, but has not restored B cell function to date. We developed a new approach for XSCID gene therapy that utilizes a safety modified lentiviral (LV) vector (CL20-i4-EF1α-hGC-OPT) together with reduced exposure busulfan (Bu) conditioning for newly diagnosed infants with XSCID. Recently, we reported that the combination of reduced dose Bu used together with our LV vector restored T and B cell function in older XSCID children with declining immune function following haploidentical transplantation (De Ravin SS et al, Sci Transl Med, 2016). Here we report initial results of LVXSCID-ND; a multi-center, phase I/ II safety and efficacy study using our LV vector and dose-adjusted Bu for the first time in newly diagnosed XSCID infants. We enrolled six subjects over the last 12 months (median age of 4.5 months, range: 2-12 months). Purified bone marrow (BM) CD34+ cells were transduced with the CL20-i4-EF1α-hGC-OPT vector generated by a stable producer cell line and then cryopreserved to facilitate central manufacturing for multiple study sites and evaluation of release criteria prior to conditioning. Busulfan was given as two single daily doses to target a total cumulative area-under-the-curve (cAUC) of 22 mg*hr/L (60-90 mg*hr/L = myeloablative cAUC). The median dose of transduced CD34+ cells was 8.06 x 106 cells/kg (range: 4.6 - 11 x 106) and the median vector copy number (VCN) in graft CFU-C was 0.40 copies /cell (range: 0.16 - 0.97). An average Bu cAUC of 22.9 mg*hr/L (range: 20.0 to 24.2) was achieved, which was within 10% of the intended cAUC in all patients. As of July 2017, no severe adverse events related to BM harvest, Bu exposure, or cell infusion have been observed. In the first 5 evaluable cases, complete hematopoietic recovery occurred by 3-4 weeks without any blood product support. Follow-up data from the oldest patient who presented with high levels of maternal T cell engraftment, severe neutropenia requiring G-CSF therapy, CMV viral infection, with a graft VCN of 0.16, and who is now 12 months post therapy, demonstrated delayed and partial T cell reconstitution. Cases 2 and 3 have been followed for nine and six months, respectively and have significantly higher VCNs in peripheral blood (PB) subsets (CD14/15+myeloid cells 0.65, 0.30 copies/cell: CD3+ T cells 2.78, 2.71; CD19+ B cells 1.01, 0.78; and CD56+ NK cells 2.72, 2.11 respectively). Bone marrow aspirates on week 16 yielded VCNs in sorted CD34+ cells of 0.56, 0.50; and BM myeloid CFU-GM of 0.61, 0.24. Rapid T cell reconstitution in cases 2 and 3 resulted in normal numbers of CD3+, CD4+, CD4 naïve, and CD8+ cells with TRECs 626 and 1170 copies per ug DNA 16-20 weeks post therapy. In both cases at 16 weeks, T cell proliferation was 86% and 81% of control, and V-β spectratype scores were normal at 194 and 155. At nine months, case 2 had a normal isohemagglutinin anti-A titer of 1:32 and normal 4-week trough serum immunoglobulins levels (IgG 713 mg/dl, IgM 54 mg/dl, and IgA 16 mg/dl). IVIG supplementation has been discontinued for approximately 3 months before vaccination responses are assessed. Case 4 has been followed for 12 weeks with PB VCNs for CD3+ cells of 1.36 copies/cell, CD19+ 0.58, CD56+ 1.38 and CD14/15+ 0.02. Flow cytometry analysis of PB at 12 weeks shows 4.5% of PB leukocytes are CD3+, with 61% CD4+, 11% CD8+, and a significant fraction expressing a CD45RA+, RO- naive phenotype at this early time point. Cases 5 and 6 are still too early to evaluate for immune phenotype and function. Preliminary vector insertion site analysis shows highly polyclonal marking patterns in case 2 with 11,000 insertion sites in CD3+ cells, 5049 sites in CD19+ cells, 756 sites in BM myeloid CFU-C without any evidence of clonal dominance. In summary, gene therapy for newly diagnosed XSCID patients using a LV vector with targeted reduced exposure Bu conditioning is well tolerated and results in rapid T cell reconstitution in most cases. Efficient vector marking in bone marrow CD34+ cells, myeloid cells, and B cells indicate that this approach will likely provide broad immune reconstitution rather than restricted T cell correction seen in past trials using γ-retroviral vectors with no Bu. DisclosuresLong-Boyle:InsightRX: Consultancy. Puck:UpToDate: Patents & Royalties: Recieve royalties to write and edit entries on primary immune disorders; InVitae, a clinical DNA sequencing company: Other: Spouse employment and stock options. Cowan:Homology Medicine: Consultancy.

Lentiviral Gene Therapy with Low Dose Busulfan for Infants with X-SCID Results in the Development of a Functional Normal Immune System: Interim Results of an Ongoing Phase I/II Clinical Study

Lentiviral Gene Therapy with Low Dose Busulfan for Infants with X-SCID Results in the Development of a Functional Normal Immune System: Interim Results of an Ongoing Phase I/II Clinical Study

Academic, Phase I/II Trial on T Cells Expressing a Second Generation, CD19-Specific Chimeric Antigen Receptor (CAR) and Inducible Caspase 9 Safety Switch for the Treatment of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) and B-Cell Non-Hodgkin Lymphoma (B-NHL) in Children

Survival rates of children with relapsed/refractory (r/r) BCP-ALL remain unsatisfactory and little progress has been made in the past 2 decades. Similarly, relapse of childhood B-NHL is usually associated with an aggressive disease and poor outcomes. Targeted immunotherapy with T-cells genetically modified to express a CD19-directed CAR showed an unprecedented antitumor efficacy, leading to the recent FDA and EMA approval of two CD19-CAR products for treatment of BCP-ALL and B-NHL. Relevant toxicities have, however, been reported, mainly related to the development of severe Cytokine Release Syndrome (CRS) and/or of neurotoxicity. At Ospedale Pediatrico Bambino Gesù (OPBG) in Rome, we developed a clinical-grade, 2nd generation, CD19-specific CAR construct, including 4.1bb as costimulatory domain and the inducible caspase-9 safety switch (iC9-CD19-CAR), vehiculated by a retroviral vector, to conduct an academic, phase I/II clinical trial in patients (age 1-25 yrs) affected by BCP-ALL or B-NHL. We now report on the results of the phase I and of the first 8 patients treated in the phase II portion of the study, in terms of feasibility, toxicity, maximum tolerated/recommended dose (MTD/RD) and data on response rate and biological correlates. The phase I, dose-escalation portion of the study included 3 dose levels (DL), namely: DL1, 0.5×106; DL2, 1.5×106; DL3, 3.0×106 CAR+ T cells per kg of recipient body weight. In the phase II portion, patients were treated at the RD identified in the phase I, namely 3.0×106 CAR+cells/kg. All patients received a lymphodepleting regimen consisting of fludarabine and cyclophosphamide for 3 days and iC9-CD19-CAR T cells were subsequently administered as single infusion. Patients were monitored for toxicity, expansion and persistence of iC9-CD19-CAR T cells. Seventeen children were enrolled into the trial and received iC9-CD19-CAR T cells between January 2018 and June 2019. Data were analyzed as of July 20, 2019. The characteristics of the patients are detailed in table 1. The designed dose concentration was successfully produced for all the enrolled patients and we did not observe any production failure. The median transduction rate in the drug product was 54% (range 21-73), while the median vector copy number was 3.8 (range 2.8-6.2). During the phase I portion of the study, no dose limiting toxicities (DLTs) have been recorded, defining the MTD as 3.0×106 CAR+ T cells per kg of recipient body weight. The treatment was overall tolerated and all the toxicities were reversible, the most severe being grade 3-4 neutropenia, thrombocytopenia and/or anemia, occurring in 16/17 (94.1%) patients; in 13/16 patients (81.2%) the hematological toxicity developed before the infusion and persisted after the administration of CAR T cells. Cytokine release syndrome (CRS) occurred in 10/17 patients (58.8%) and was overall moderate, reaching grade 3 (Lee criteria) in one patient only. Notably, none of the patients developed neurotoxicity and no activation of the safety switch was required. All patients were assessed for response at 4 weeks from iC9-CD19-CAR T cell infusion and 13/15 (86.7%) patients with ALL achieved complete remission (CR) with negativity of minimal residual disease (MRD), including 2/3 patients receiving the DL1, 9 patients who had failed a previous allogeneic haematopoietic stem-cell transplantation (HSCT) and 6 patients that had previously received blinatumomab, as CD19-directed immunotherapy. The iC9-CD19-CAR T cells expanded in vivo and were detectable by both flow-cytometry and molecular biology in the blood (Fig.1), bone marrow and cerebrospinal fluid of the responders. One CD19-negative relapse 3 months after infusion was recorded, while 3 additional patients relapsed with CD19+ leukemia blasts. Four patients received HSCT while in CR with MRD negativity because of regrowth of normal CD19+ B cells. The 18-month probability of overall survival for the BCP-ALL cohort is 72.2% (Fig.2). One of the 2 B-NHL patients showed a partial response. Our data indicate that iC9-CD19-CAR T cell in an academic setting is feasible, safe and extremely effective in treating highly resistant/relapsed BCP-ALL. In our trial, no major or life-threatening toxicities were observed and, despite the moderate CRS recorded, high rates of CR were achieved, suggesting that the combination of a retroviral platform and 4.1bb as costimulation is able to mediate a potent antitumor effect Disclosures Merli: Amgen: Honoraria; Novartis: Honoraria; Sobi: Consultancy; Bellicum: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Miltenyi: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

Clinical-Grade Transduction of Allogeneic Cytokine Induced Killer (CIK) Cells with CD19 Chimeric Antigen Receptor (CAR) Using Sleeping Beauty (SB) Transposon: Successful GMP-Compliant Manufacturing for Clinical Applications

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). [Display omitted] DisclosuresGritti:Autolus: Consultancy. Rambaldi:Celgene: Consultancy; Omeros: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy; Pfizer: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy.

Lentiglobin Gene Therapy for Transfusion-Dependent β-Thalassemia: Update from the Northstar Hgb-204 Phase 1/2 Clinical Study

Lentiglobin Gene Therapy for Transfusion-Dependent β-Thalassemia: Update from the Northstar Hgb-204 Phase 1/2 Clinical Study

Interim Results from a Phase 1/2 Clinical Study of Lentiglobin Gene Therapy for Severe Sickle Cell Disease

Interim Results from a Phase 1/2 Clinical Study of Lentiglobin Gene Therapy for Severe Sickle Cell Disease