SIN Lentiviral Vector Research Articles

Automated Manufacture of CAR19 Donor Lymphocytes Incorporated into an Escalated Dose DLI Protocol: Preliminary Results from the CARD Study

Introduction:Clinical studies of 2nd generation CD19 Chimeric Antigen Receptor (CAR) T-cells have delivered unprecedented clinical responses in patients with B-ALL. Increasing demand for CAR T-cells places huge pressure on the small number of laboratories with expertise in GMP CAR T-cell manufacture. Automation of CAR T-cell production using the CliniMACS Prodigy (Miltenyi Biotec) has enormous potential to simplify manufacture and to enhance CAR T-cell accessibility to patients.We have optimized and successfully validated GMP-grade CD19CAR T-cell manufacture on the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cells in the Phase I CARD study (NCT02893189). Recruitment to the CARD study is open to adult patients with relapsed CD19+ disease following allogeneic stem cell transplantation (allo-SCT). The study is designed to assess the feasibility of automated CD19CAR T-cell manufacture and to assess the engraftment and safety profile of allogeneic CD19CAR T-cells generated using the CliniMACS Prodigy.Methods:To validate automated CAR T-cell manufacture on the CliniMACS Prodigy, large-scale experiments using cryopreserved or fresh healthy donor leukapheresis were performed and assessed for T-cell expansion, transduction efficiency, viability, purity, immunophenotype and functionality. T-cells were activated with TransAct, a polymeric nanomatrix incorporating CD3/CD28-specific antibodies, transduced with a SIN lentiviral vector encoding a second generation CD19 CAR (4G7-41BBz CAR) and underwent expansion in a closed system with automated feeding and media exchange prior to cryopreservation. Small-scale controls with TransAct / TexMACS or Dynabeads / X-vivo were run in parallel.The CARD study is designed to mirror escalated dose DLI protocols, allowing up to 3 consecutive doses of CD19CAR T cells at intervals of no shorter than 8 weeks, titrated to clinical response. Cell doses are based on CD3+ T-cell numbers rather than on absolute CD19CAR T-cell numbers (dose 1 = 1x106/kg CD3+ T-cells; dose 2 = 3x106/kg CD3+ T-cells and dose 3 = 1x107/kg CD3+ T-cells). A minimum transduction efficiency of 10% is stipulated as a release criterion for all trial products.Results:Preclinical scale up studies of the automated CAR T-cell manufacturing process with healthy donor leucapheresate confirmed equivalence of CAR T-cell specification and yield when compared to products manufactured using established WAVE-Bioreactor-based GMP T-cell processes in our facility (Qasim et al, Cytotherapy, 2016). Based on this data, automated CliniMACS Prodigy-based CD19CAR T-cell manufacture was subsequently approved by the MHRA, the UK regulatory body, for use in the CARD clinical trial.To date, we have enrolled 3 patients on study. Transduction efficiency was on average 46.6% (range 40.7 - 55.3%), with successful manufacture of 3 CD19CAR T-cell doses per patient. All products met sterility release criteria. GMP capacity, staffing and quality assurance systems permit manufacture of one CliniMACS Prodigy product per month using a team of 2 GMP staff with the possibility for further upscaling. Two patients have been treated at the initial T cell dose, with evidence of engraftment and expansion despite the absence of preconditioning.Conclusions:We have optimized and successfully validated GMP-grade CD19CAR T-cell manufacture using the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cell products to adult patients with CD19+ disease relapse following allo-SCT. Preliminary results of CAR T-cell manufacture and clinical outcomes in initial patients will be presented. DisclosuresNickolay:Miltenyi Biotec GmbH: Research Funding. Mock:Miltenyi Biotec GmbH: Research Funding. Himoudi:Autolus: Research Funding. Thrasher:Orchard Therapeutics: Consultancy; Torus Therapeutics, Inc: Other: Advisory Board; Rocket Pharmaceuticals Ltd: Consultancy; 4bio Ventures Management Ltd: Other: Advisory Board. Pule:UCL: Patents & Royalties: UCL Business; Autolus Ltd: Employment, Equity Ownership, Research Funding. Qasim:Miltenyi: Research Funding; Servier: Research Funding; NIHR: Research Funding; Autolus Ltd: Consultancy, Equity Ownership; Bellicum: Research Funding; Cellectis: Research Funding; Orchard Therapeutics: Consultancy, Equity Ownership.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Universal' Fratricide-Resistant CAR T Cells Against T Cell Leukemia Generated By Coupled & Uncoupled Deamination Mediated Base Editing

Chimeric antigen receptor (CAR) T cell approaches to target T cell malignancies have been hampered by the fundamental issue of 'T v T' fratricide when T lineage antigens such as CD3 or CD7 are targeted. Genome-editing can be employed to efficiently eliminate expression of shared target antigens, and multiplexed approaches can deliver simultaneous removal of additional molecules relevant to generating 'universal' T cells, such as the αβ T cell receptor, β2m and CD52, the target antigen for Alemtuzumab. However, these strategies have generally relied on nuclease mediated double stranded DNA cleavage and repair by non homologous end joining (NHEJ), and this can trigger apoptosis and generate predictable and unpredictable chromosomal translocations. Base editing using CRISPR guided chemical deamination offers the possibility of highly precise, seamless, cytidine to uridine deamination (resulting in C→T or G→A substitutions) which can be directed to create stop codons or to disrupt splice donor/acceptor sites. This enables simultaneous genetic disruption of critical antigens or receptors required for the generation of 'off the shelf' cell banks that can be infused in combination without fratricidal effects. A CRISPR-CAR coupled lentiviral configuration with a single guide RNA (sgRNA) specific for the constant region of TCR β chain (TRBC) was incorporated into the 3'ΔU3LTR of the vector under the control of a minimal U6 pol III polymerase promoter. The configuration supported high level base conversion and efficiently disrupted TCRαβ expression. When followed by stringent magnetic bead mediated depletion of residual TCRαβ T cells, highly homogenous CAR+TCR- populations with <1% residual TCRαβ T cells were obtained, an important consideration in the application of mismatched allogeneic T cells. Combinational delivery during electroporation of additional, uncoupled sgRNAs against additional molecules relevant to overcoming HLA barriers supported up to 92% triple knockout of TCR/CD52/β2M in CAR expressing cells. CRISPR guide RNAs targeting CD7 and/or TRBC (for TCR/CD3 disruption) were delivered by electroporation to primary T cells in combination with codon-optimized (co) base editor 3 (BE3) or coCas9 mRNA, ahead of transduction with sin-lentiviral vectors expressing CARs against CD3 or CD7. Simultaneous disruption of TCR/CD3 and CD7 was confirmed by flow cytometry and verified by direct sequencing of the target loci. Efficient multiplexed editing enabled co-culture of T cell populations expressing anti-CD3 and anti-CD7 specific CARs, with fratricidal elimination of residual non-edited populations. Chromium release and flow based cytotoxic functional responses were similar between cells generated using coCas9 and coBE3. However, digital droplet PCR of coCas9 edited cells detected low frequency (1-3%) predicted translocation events, while these were barely detectable in base edited cells. In vivo anti-leukemic functionality of base edited CAR T cells was verified by serial imaging of NOD/SCID/γc mice engrafted with luciferase labelled Jurkat T cells modified to express CD3, or CD7, alone or in combination. Effector inoculation with T cells expressing anti-CD3 and anti-CD7 CARs inhibited leukemic expansion and luciferase signal. Additional characterisation of T cells following multiple deamination mediated editing is underway to investigate wider RNA and DNA effects of chemical deamination. The strategies are readily scalable through the adaption of existing semi-automated manufacturing processes. A time limited therapeutic application of 'universal' anti-T CAR T cells is planned to deliver leukemic clearance and deep molecular remissions ahead of conditioning and programmed allogeneic stem cell transplantation for donor derived multilineage immune reconstitution. Disclosures Qasim: UCLB: Other: revenue share eligibility; Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding.

Early Clinical Experience of CD19 x CD22 Dual Specific CAR T Cells for Enhanced Anti-Leukemic Targeting of Acute Lymphoblastic Leukemia

Introduction:Advances in chimeric antigen receptor (CAR) T cell therapy have yielded complete remission (CR) rates in relapsed/refractory B-ALL (rrB-ALL) of 70-95%. However, disease recurrence after CD19 or CD22 CAR therapy is greater than 50% at 1 year, and approximately half of recurrences are due to antigen escape. To reduce antigen escape and optimize the durability of remission, we sought to design a CAR T cell product with dual specificity that is capable of simultaneously targeting both CD19 and CD22. Preclinical testing of our bi-specific CAR showed a preference for signaling through CD22 over the CD19 CAR. In contrast, dual transduced T cells signaled through both the CD19 and CD22 CAR with lytic activity and cytokine production similar to single transduced CAR T cells of the same specificity. Therefore, we opted to move forward with dual transduced T cells for clinical use. We are currently testing SCRI-CAR19x22v1 in PLAT-05 (NCT03330691), a phase 1 clinical trial for pediatric and young adult patients with CD19+CD22+ rrB-ALL, with the primary objectives to determine the feasibility of manufacturing products with dual specificity, to assess the safety of the cryopreserved product infusion, and to describe the full toxicity profile.Methods:Subjects undergo apheresis, after which the CD4 and CD8 T cell subsets are immunomagnetically selected and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two separate SIN lentiviral vectors that direct the expression of a CD19-specific FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR with an Her2tG tag and expression of a CD22-specific m971scFv:IgG4hinge:CH2(L235D)-CH3:CD28tm:4-1BB:ζ CAR with an EGFRt tag, creating three distinct populations of CAR T cells (anti-CD19, anti-CD22, and anti-CD19x 22). Transduced cells are expanded in serum free media formulation with IL-7, IL-15, and IL-21. Following lymphodepleting chemotherapy, cryopreserved products are thawed and infused at the protocol-prescribed dose level. Cytokine release syndrome (CRS) is graded according to Lee et al. (Blood 2014) and is treated according to our early intervention strategy of tocilizumab and dexamethasone for persistent, mild CRS.Results:Seven subjects (ages 1-26 yr) with rrB-ALL have been enrolled with 4 treated at dose level 1 (1 x 106 CAR T cells/kg) and 3 treated at dose level 2 (3 x 106 CAR T cells/kg). The mean culture time was 7.9 days (range 7-11) and subjects received infusions with a mean CD8:CD4 ratio of 1.7 (range 0.2 - 3.1). CD8 CAR composition, on average, consisted of 21.6 % CD19 CAR, 37.8 % CD22 CAR, and 40.6 % CD22xCD19 CAR T cells. CD4 CAR composition, on average, consisted of 25.8 % CD19 CAR, 30.6 % CD22 CAR, and 43.6 % CD22xCD19 CAR T cells (Figure). Peak engraftment occurred between days 7 and 14 for all patients and was predominantly composed of the CD19 CAR population with median peak values for CD19 CAR, CD22 CAR, and CD19xCD22 CAR T cell populations of 9.1%, 1.2%, and 2.4%, respectively. A CR was achieved in 5/7 (71%) subjects by day 21, 4 of which were minimal residual disease negative. The two subjects without a CR did not exhibit evidence of CAR T cell engraftment; one had previously received CD19 CAR T cells, and the other had progressive disease and pursued alternative therapy at day 10. Therapy was well tolerated with no dose limiting toxicities. CRS occurred in 5 subjects (Grade 1) with 2 of these subjects experiencing mild neurotoxicity (Grade 1). Four subjects received tocilizumab +/- dexamethasone, and two of these received multiple doses of dexamethasone.Conclusions:Preclinical testing showed superior efficacy against both CD19 and CD22 when using two separate CARs and dual transduction, compared to a single bi-specific CAR. Preliminary analysis of PLAT-05 supports feasibility of product manufacturing, and toxicity and response rates that are consistent with the reported CD19 CAR T cell experience. While the infused SCRI-CAR19x22v1 products consist of a near-uniform distribution of the 3 distinct populations, we observed selective in vivo expansion of the CD19 CAR T cell population. Further investigation is required to understand the mechanism of CD19 CAR dominance in vivo. Continued accrual of subjects is ongoing to further assess the impact of dual antigen targeting on the prevention of antigen escape and the potential to provide a more durable remission. [Display omitted] DisclosuresPark:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Early Response Data for Pediatric Patients with Non-Hodgkin Lymphoma Treated with CD19 Chimeric Antigen Receptor (CAR) T-Cells

Background:Pediatric patients with relapsed or refractory CD19+non-Hodgkin lymphoma (NHL) have poor outcomes despite use of chemotherapy and hematopoietic stem cell transplant (HSCT). Clinical trials of CD19 CAR T-cells have demonstrated efficacy in salvaging adult patients with relapsed and refractory NHL.Objectives:The objectives of this analysis is to assess the safety, toxicity, feasibility and efficacy of SCRI-CAR19v1 for pediatric patients with relapsed or refractory NHL.Design/Methods:The ongoing phase 2 trial (NCT02028455) has enrolled and treated 8 pediatric subjects with CD19+NHL. Subjects underwent apheresis, with their CD4 and CD8 T cell subsets prepared immunomagnetically. T cells were stimulated with anti-CD3xCD28 bead stimulation, and then transduced with a SIN lentiviral vector to direct co-expression of the FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR and the selection/tracking/suicide construct EGFRt. The transduced cells were propagated using recombinant human cytokines to numbers suitable for clinical use. Subjects received lymphodepletion of fludarabine and cyclophosphamide followed by 1x106CD19 CAR T-cells/kg as a 1:1 ratio of CD4 and CD8 cells. Response was assessed at 3 and 9 weeks. Adverse events were graded according to CTCAEv4 except cytokine release syndrome (CRS) was graded according to Lee et al.Results: Treated subjects had relapsed or refractory diffuse large B cell lymphoma (DLBCL) (4/8), Burkitt's lymphoma (2/8), gray zone B cell lymphoma (1/8), primary mediastinal B cell lymphoma (PMBCL) (1/8), and ranged from 4-18 years old. Two subjects received prior hematopoietic stem cell transplant (HSCT); the subject with PMBCL received auto- and allogeneic HSCT and a subject with Burkitt's received autologous HSCT. Five subjects received prior immunotherapy with brentuximab, nivolumab, rituximab, and/or obinutuzumab. One subject had received ibrutinib. No subject had received prior CAR T-cells. CD4 and CD8 products were successfully manufactured and infused for all subjects. All subjects had expansion of CAR T-cells in the peripheral blood, bone marrow and CSF, with ongoing persistence at last check (range 14 days - 9 months). Toxicity information through day 30 revealed the occurrence of mild CRS in 4 subjects (grade 1 n=3, grade 2 n=1), and one case of severe CRS (grade 3). Mild neurotoxicity was observed in 2 subjects (grade 1 n=1, grade 2 n=1) with no occurrence of severe neurotoxicity. Response assessment at 3 weeks (n=6) revealed anti-tumor responses in 5 subjects, including complete response (CR) by week 9 (n=2, both DLBCL). CR was not sustained in either subject despite ongoing CAR T cell persistence. One of these subjects had a PET avid lesion proven by biopsy to be necrotic tissue but subsequently developed CD19+recurrence at that site. The other subject developed a new CD19+site of disease at six months; however, achieved a 2nd CR 3 weeks after receiving a second infusion of the originally manufactured CAR T-cells. One partial response (PR) subject experienced clearance of marrow disease with stable lymphoma but developed CD19 negative progression at 9 weeks. Updated enrollment, toxicity and response assessments will be presented.Conclusion:SCRI-CAR19v1 therapy demonstrates efficacy in pediatric patients with relapsed and refractory NHL and appears to be well tolerated with less severe toxicities than observed for pediatric patients with CD19+leukemia. Persistence of the CAR T-cells is excellent with no early loss of CAR T-cell engraftment reported to date. Although early responses were observed, these were not durable perhaps reflecting biologic/immunologic differences between B cell lymphomas in children in comparison to NHL in adults. DisclosuresPulsipher:Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria; CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Park:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Impaired X-CGD T cell compartment is gp91phox-NADPH oxidase independent

Chronic granulomatous disease (CGD) is a phagocytic disorder characterized by a defective production of reactive oxygen species (ROSs). Although infections and granuloma formation are the most common manifestations in CGD patients, a significant number of patients experienced autoimmunity and inflammatory diseases suggesting that adaptive immune abnormalities might be involved.Here we investigated T-cell compartment and showed that CGD patients had a skewed TCRV-beta distribution in CD8+ T cells, particularly in older patients, and a reduced proliferative responses toward mitogens compared to healthy donors (HD). Afterwards we studied the role of gp91phox protein in causing these alterations and demonstrated that human T cells do not express gp91phox and TCR-stimulated ROS generation is gp91phox-NADPH oxidase independent. Finally, we proved that the NADPH oxidase is not active in the T cell compartment even when forcing gp91phox expression transducing T cells from X-CGD and HD with a SIN lentiviral vector (LVV) encoding the gp91phox cDNA.

A Novel Low Affinity CD19CAR Results in Durable Disease Remissions and Prolonged CAR T Cell Persistence without Severe CRS or Neurotoxicity in Patients with Paediatric ALL

Introduction:Published studies of CD19 CAR T cells have shown unprecedented response rates in ALL but with a 23-27% incidence of severe Cytokine Release Syndrome (CRS) and 27-50% incidence of severe neurotoxicity which may limit broader application. We developed a novel second generation CD19CAR (CAT-41BBz CAR) with a lower affinity and faster off-rate but equivalent on-rate than the FMC63-41BBz CAR (Kd 116 nM vs 0.9 nM, T1/2 10s vs 1260s) utilised in CTL019 currently under consideration by the FDA. Pre-clinical studies indicated T-cells transduced with CAT-41BBz mediate enhanced tumor clearance and show increased expansion in an NSG-NALM6 stress test model (Kramer et al., submitted). We here report interim results from a multi-centre, Phase I clinical study of autologous CAT-41BBz CAR T cells as therapy for high risk/relapsed paediatric ALL, CARPALL (NCT02443831) demonstrating efficacy with an excellent safety profile.Methods:Autologous T cells were activated with anti-CD3/CD28 beads, transduced with a SIN lentiviral vector encoding CAT-41BBz CAR and expanded for 4 days prior to magnetic bead removal and cryopreservation. Transduction efficiency was assessed using an anti-idiotype antibody. Serum levels of cytokines associated with CRS were measured using cytometric bead array.All patients received lymphodepletion with fludarabine 150 mg/m2 + cyclophosphamide 1.5g/m2 followed by a single infusion of CAR T cells at a dose of 1x106 CAR+ T cells. Patients were monitored for the presence of CAR T cells in the blood by flow cytometry and by qPCR for the 41BBz junctional region, as well as circulating B cell count monthly for 6 months and then 6 weekly to 1 year. Disease status was assessed in the bone marrow morphologically, by IgH qPCR, as well as by flow cytometric assessment of MRD at the same time-points to establish durability of responses as a stand-alone therapy. The primary end-points were incidence of grade 3-5 toxicity related to CAR T cells within 30 days and the proportion of patients achieving molecular remission.Results:We have enrolled 10 patients and treated 8 to date. Six of 8 had relapsed post myeloablative SCT. The median disease burden prior to lymphodepletion was 9% blasts (ranging from molecular CR to 74% blasts, Table 1). It was possible to generate a product meeting release criteria in all but 1 patient (90% feasibility). Median transduction efficiency was 18.1% (range 6.7 to 76.3%). All treated patients received the anticipated dose of 1x106 CAR T cells/kg.Cytokine release syndrome occurred in all patients (grade 1 n=4, grade 2 n=4), but to date none have developed ≥ grade 3 CRS, required ICU admission or therapy with Tocilizumab. CRS was associated with modest elevations of IL-6, IFN-γ and IL-10 and resolved spontaneously in all. Grade 2 neurotoxicity was observed in 3 patients and resolved spontaneously, but no severe (≥grade 3) neurotoxicity was seen. Five patients had prolonged grade 4 neutropenia lasting > 30 days but this resolved in all by 2 months. Only 1 patient experienced significant infective complications in the context of pre-existing poor marrow reserve following allogeneic SCT.6/7 (86%) evaluable patients achieved molecular remission at a median of 30 days post infusion (range 30-60 days, Table 1). One patient did not respond and died of CD19+ disease progression. At a median follow-up of 5.9 months (range 28-328 days), 4/7 evaluable patients remain in flow MRD negative remission of whom 3 show no evidence of molecular MRD at 1, 7.5 and 9 months. Two patients relapsed with CD19- disease at 3 and 4 months post infusion: 1 of these remains alive with disease at 11 months and the other died of disease progression.Reflecting our pre-clinical data with CAT-41BBz CAR, we have seen excellent CAR T cell expansion (median 65459 copies/µg DNA at 1 month, range 609 to 230112) and persistence at up to 11 months post-infusion (Figure 1). All 7 evaluable patients have ongoing CAR T cell persistence detectable by both flow and qPCR as well as ongoing B cell aplasia at last follow-up.Conclusions:These interim results with a novel low affinity CD19 CAR show similar remission rates to those reported by US studies in paediatric ALL with an improved safety profile. No severe (grade ≥3) CRS or neurotoxicity has occurred to date despite high tumour burden in 4 patients. Excellent CAR T cell expansion has been documented, as well as long duration of CAR T cell persistence and associated B cell aplasia. [Display omitted] DisclosuresGhorashian:UCL: Patents & Royalties: UCL Business. Kramer:UCL: Patents & Royalties: UCL Business. Lucchini:Alexion: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL: Patents & Royalties: UCL Business.

A Review of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any of the five subunits of the NADPH oxidase complex responsible for the respiratory burst in phagocytic leukocytes. Patients with CGD are at increased risk of life-threatening infections with catalase-positive bacteria and fungi and inflammatory complications such as CGD colitis. The implementation of routine antimicrobial prophylaxis and the advent of azole antifungals has considerably improved overall survival. Nevertheless, life expectancy remains decreased compared to the general population. Inflammatory complications are a significant contributor to morbidity in CGD, and they are often refractory to standard therapies. At present, hematopoietic stem cell transplantation (HCT) is the only curative treatment, and transplantation outcomes have improved over the last few decades with overall survival rates now > 90% in children less than 14 years of age. However, there remains debate as to the optimal conditioning regimen, and there is question as to how to manage adolescent and adult patients. The current evidence suggests that myeloablative conditioning results is more durable myeloid engraftment but with increased toxicity and high rates of graft-versus-host disease. In recent years, gene therapy has been proposed as an alternative to HCT for patients without an HLA-matched donor. However, results to date have not been encouraging. with negligible long-term engraftment of gene-corrected hematopoietic stem cells and reports of myelodysplastic syndrome due to insertional mutagenesis. Multicenter trials are currently underway in the United States and Europe using a SIN-lentiviral vector under the control of a myeloid-specific promoter, and, should the trials be successful, gene therapy may be a viable option for patients with CGD in the future.

New Molecular Surrogate Assay for Genotoxicity Assessment of Gene Therapy Vectors (SAGA)

The variety of gene therapy vectors for a multitude of different diseases has increased tremendously over the years. However, a number of patients that underwent gene therapy in different trials developed hematological malignancy caused by integration of the provirus in the vicinity of proto-oncogenes. These severe adverse advents prompted intense research efforts towards safer gene therapy, leading to the removal of the long terminal repeat enhancer elements and the use of internal promoters in retroviral vectors. Still, a bottleneck of transition from basic research to clinical application is the test for safety of integrating retro- and lentiviral vectors.Instead of laborious in vivo models with limited predictive value, in vitro assays to screen for insertional mutagenesis are strongly desirable. A decade ago, our lab developed the in vitro immortalization (IVIM) assay to quantify the genotoxic potential of viral vectors, which has been widely used to complete preclinical safety documentation of newly developed integrating vector systems. Despite general acceptance in the field of hematopoietic gene therapy, bias for insertional mutants of the myeloid lineage, a low sensitivity and a long assay run time are clear limitations.We now developed the molecular surrogate assay for genotoxicity assessment (SAGA). The new test is more robust, sensitive and biologically informative. As input we used murine lineage-negative hematopoietic stem and progenitor cells (HSPC) that were cultured as described for the IVIM assay. The murine HSPC were transduced with a number of different gammaretro- and lentiviral vectors, including vectors that have been employed in clinical trials for X-SCID and Wiskott-Aldrich Syndrome. After 14 days, whole mRNA was isolated from transduced and non-transduced samples and analyzed by Agilent custom microarrays (n=86) and qPCR from nine independent SAGA assays. We applied several Machine Learning algorithms to derive a core set of genes which distinguishes transformed from non-transformed samples in each individual SAGA assay. This set of genes from the individual analysis was further analyzed to derive a core set of genes that is able to robustly separate transformed from non-transformed samples in all assays performed. In order to account for platform-specific effects we validated all microarray results by conventional qPCR-methodology. The SAGA gene set was then cross-validated in an independent validation cohort of SAGA-assays that were not part of the SAGA-training set from which the signature was derived from.The SAGA assay was used to quantify the mutagenic potential of several benchmark vectors. It correctly assigned a high mutagenic potential to vectors (MFG.yc and CMMP.WASP) which led to serious adverse events (SAEs) in clinical trials. Most importantly, the SAGA assay reliably scored high for mutagenic vectors, even when the vector did not transform in IVIM-assays conducted in parallel, demonstrating the higher sensitivity of the SAGA-principle. In contrast, SIN lentiviral vectors with weaker internal promoters (LV.EFS.yc and LV.EFS.ADA) showed no enrichment of the SAGA-core signature and hence scored much safer in the SAGA test. We present the results for these vectors side-by-side either using IVIM or SAGA. In summary, we generated an advanced version of the currently used in vitro insertional mutagenesis screening system by integrating a molecular read-out which enhances reproducibility, sensitivity and reduces assay duration, paving the way for a better preclinical risk assessment of gene therapy vectors. DisclosuresNo relevant conflicts of interest to declare.

CD19CAR T Cell Products of Defined CD4:CD8 Composition and Transgene Expression Show Prolonged Persistence and Durable MRD-Negative Remission in Pediatric and Young Adult B-Cell ALL

▪Introduction:Multiply recurrent pre-B-cell ALL, and particularly relapse following allo-HSCT, has dismal outcomes due in large part to ineffectual therapies. The primary objectives of the Phase 1 portion of the PLAT-02 study (NCT02028455) were to determine the feasibility of manufacturing products of defined composition and transgene expression, the safety of the cryopreserved T cell product infusion, and to describe the full toxicity profile, including development of clinically significant GVHD in the post-allo-HSCT cohort.Methods:Subjects on the PLAT-02 study undergo apheresis, with their CD4 and CD8 T cell subsets prepared immunomagnetically. Following anti-CD3xCD28 bead stimulation, T cell lines are transduced with a SIN lentiviral vector that directs the co-expression of the FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR and the selection/tracking/suicide construct EGFRt. Transduced cells are propagated using recombinant human cytokine cocktails to numbers suitable for clinical use over 10-22 days, during which time they are subjected to EGFRt immunomagnetic positive selection. Shortly following lymphodepleting chemotherapy, cryopreserved CD4/EGFRt+ and CD8/EGFRt+ T cell products are thawed and infused at the bedside such that patients receive a 1:1 ratio of EGFRt+ CD4 and CD8 T cells at the protocol-prescribed dose level.Results:45 subjects have been enrolled and 43 have been treated from dose level 1 (5 x 105 CAR-T cells/kg) through 4 (10 x 106 CAR-T cells/kg). Therapeutic T cell products were released on all 45 enrolled subjects, with 1 subject requiring a second apheresis. Two subjects died of disease prior to their infusion. All 43 infused subjects received lymphodepletion chemotherapy prior to T cell infusion (cyclophosphamide, n=27; fludarabine/cyclophosphamide; n=14 cyclophosphamide/etoposide n=1; fludarabine n=1,). 91% (39/43) of subjects received infusions at the desired 1:1 CD4:CD8 ratio and their infusions were well tolerated with only 1 related AE >grade 2. 93% (40/43) of subjects had a documented MRD-negative CR within 21 days following CAR-T cell therapy. The 12 month event-free survival (EFS) is 50.8% (95% CI 36.6, 69.9) and 12 month OS is 69.5% (95% CI 55.8, 86.5). All responding subjects exhibited in vivo expansion of CAR-T cells. The % of CAR T cell expansion over time is not impacted by dose level or lymphodepletion but is impacted by disease burden (p=0.004) and total CD19 antigen burden (p=0.001) at the time of lymphodepletion. The median duration of functional CAR-T cell persistence as measured by ongoing B-cell aplasia (BCA) is impacted by the total CD19 antigen burden in the bone marrow at time of lymphodepletion (>15% vs <15%), with a median of 6.4 months vs 1.7 months (p=0.005), respectively. Flu/cy may also affect the duration of BCA with a median of 6.4 months vs 2.1 months for alternative lymphodepletion (p=0.15). Of the 18 relapses, 7 were CD19 negative. Loss of functional persistence of CAR T cells is associated with CD19+ relapse with a HR of 34 (95%CI 2.1, 552; p=0.013). Any grade CRS was seen in 93% (40/43) of infused subjects with severe CRS in 23% (10/43). Any grade neurotoxicity was seen in 49% (21/43) with a rate of severe neurotoxicity of 21% (10/43). Severity of CRS was only related to dose level (p=0.032), with no significant difference based on disease burden, total CD19 antigen burden or lymphodepletion regimen. Severity of neurotoxicity was only related to the occurrence of severe CRS (p=0.016). There were no toxic deaths. The recommended phase 2 dose is 1 X 106 CAR-T cells/kg with flu/cy conditioning.Conclusions:Infusions of defined composition CD4:CD8 CD19 CAR/EGFRt+ T cells/kg produce high rates of MRD-negative CR in pediatric and young adult B-cell ALL patients. Based on intent to treat analyses without a proliferation screen, we have found it is feasible to generate CAR products from each of the enrolled subjects. Despite the high rates of MRD-negative CR, the durability of remission is highly influenced by the functional persistence of CAR-T cells. Strategies to enhance persistence are currently being investigated, including episodic antigen stimulation through subject derived tAPC engineered to express truncated CD19. DisclosuresGardner:Amgen: Honoraria. Li:Juno Therapeutics: Employment, Equity Ownership. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

225. Preserving CD4+ T-Cells Phenotype and Function Upon Ex Vivo Lentiviral Transduction

Ex vivo gene modification of T-cell is an appealing strategy for the treatment of both immunodeficiencies and neoplasia. Cell activation remains an essential prerequisite to genetic modification by lentiviral transduction. However, most current protocols for T-cell activation alter significantly the phenotype of transduced cells. This phenotypic change can constitute a drawback when the gene modification aims to correct a gene defect while preserving the T cell-subsets diversity. As we plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma where the patients will receive autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells, we investigate the use of the TransAct™ reagent (a CD3/CD28 nanoscale activator reagent from Miltenyi Biotec, Bergisch Gladbach, Germany) for the clinical grade transduction of CD4+ T-cells with the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an efficient transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, after activation with the TransAct™ reagent, ex vivo transduced CD4+ T-cells display very few changes in their surface markers with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 50% of cells suggesting a more “gentle “ activation of the transduced CD4+ T-cells. Phenotypic analysis of transduced cells show no overt modification of cell subsets containing TH1, TH2 and TH17 memory T-cells. Furthermore, transduced T-cells retained the ability to be primed towards the TH1, TH2 and TH17 pathways. Cell sorting of ex vivo modified cells demonstrate a preferential transduction of naive CD31+CCR7+CD62L+CD45RA+ T-cells as compared to other subsets. Besides, we did not find any significant impact of the transduction process on the TCRβ repertoire diversity as evaluated by high-throughput sequencing and analysis of diversity through the Gini-Simpson index or the Shannon index. This optimized protocol could be of valuable use for clinical trials aiming to provide the patient with diversified gene-corrected T-cells.

766. Genetic Correction of Ifnγr1 Deficiency in Hematopoietic Cells Repairs the Cellular Phenotype of Mendelian Susceptibility to Mycobacterial Disease

Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare primary immunodeficiency characterized by recurrent severe infections with otherwise only weakly virulent mycobacteria. MSMD is associated with mutations in different genes, all leading to an impaired activation of macrophages by T-cells and a defective innate immune response. Specifically mutations in the interferon-gamma (IFNγ)-receptor-1 or-2 (IFNγR1 /2) genes result in a life-threatening disease phenotype with most patients dying in early childhood. We here introduce a novel gene therapy approach for IFNγR1 deficiency. Thus, we have designed a 3rd generation SIN lentiviral vector expressing the murine cDNA of Ifnγr1 from a spleen focus forming virus (SFFV) promoter element coupled by an IRES to GFP (Lv.SFFV.Ifnyr1.iGFP). Transduction of hematopoietic stem/progenitor cells from Ifnγrl−/− mice showed expression of Ifnγr1 by FACS and qRT-PCR and no abnormalities in clonogenic growth when compared to WT control cells. Moreover, differentiation of transduced cells towards macrophages (MΦ) by M-CSF was normal as determined by morphology on cytospins and surface marker expression of CD11b, CD200R, CD115, CD45 and F4/80. When subjecting corrected MΦ to stimulation with IFNγ, corrected cells were able to build a functional IFNγR1-Ifnγ-IFNγR2 and internalize IFNγ as efficiently as WT cells, as suggested by the clearance of IFNγ from the medium within 24h of stimulation. Consequently, Lv. SFFV. Ifnγr1.iGFP corrected MΦ revealed restored up-regulation of HLA-DR and CD86 (B7.2) comparable to WT-MΦ. IFNγ dependent T-cell activation was evaluated using T cell receptor- transgenic T cells recognizing ovalbumin (OVA). Both WT and corrected MΦ were unable to activate T-cells in the presence of IFNγ and OVA, whereas MΦ from Ifnγr1−/− mice induced significantly stronger proliferation of T cells. This observation was accompanied by the induction of indoleamine 2,3-dioxygenase (IDO) in WT and corrected MΦ, suggesting that IDO enables MΦ to deplete tryptophan from the medium and interferes with T-cell proliferation. Finally, Ifnγrl downstream signaling showed restored phosphorylation of STAT1 in corrected MΦ, consistent with induction of iNos and Irf1 upon stimulation with IFNγ. Moreover, correction of Ifnγrl−/− MΦ led to a significantly improved anti-mycobacterial activity as measured by comparable killing of Mycobacterium Avium in corrected and WT MΦ. Thus, we here introduce a novel gene therapy approach for MSMD in the context of Ifnγr1 deficiency.

234. Efficient Generation of Stable Genetically Modified Human iPSC-Derived Macrophages for Innovative Gene and Cell Therapeutic Strategies

Hematopoietic differentiation of human induced pluripotent stem cells (iPSC) holds great promise for disease modeling, drug testing, and in particular for the development of novel gene and cell therapeutic strategies. In the past, interest has been directed primarily at the generation of reconstituting hematopoietic stem cells, a cell type as of yet problematic to obtain from hPSCs. Recently however, also long-lived mature myeloid cells such as tissue resident macrophages have been described and further characterized. Therefore, transplantation of macrophages may serve as an innovative treatment approach in several diseases such as pulmonary alveolar proteinosis (PAP) and adenosine deaminase deficiency (ADA). In this line, we have established an efficient, embryoid body-based human iPSC-differentiation protocol employing IL-3 in combination with M-SCF to continuously (up to 6 months) generate large numbers (5-10×106/week/6 well plate) of >95% pure monocyte/macrophages (iPSC-MΦ) via an intermediate “myeloid cell forming complex”. The iPSC-MΦ revealed typical morphology, surface phenotype and functionality comparable to their in vivo derived counterparts. In order to evaluate the suitability of this protocol also for gene therapy, we transduced human iPSC with 3rd generation SIN lentiviral vectors employing a minimal and safety optimized ubiquitous chromatin opening element (CBX3-UCOE) to prevent differentiation-associated epigenetic silencing. Here, GFP reporter gene expression driven by the elongation factor 1α short (EFS) promoter resulted in >90% GFP+ iPSC-MΦ after myeloid differentiation. In contrast, the EFS promoter without the CBX3-UCOE was subject to massive epigenetic silencing. Moreover, also a myeloid-biased myeloid-related protein 8 (Mrp8) promoter was efficiently protected from silencing, whilst tissue specificity was not affected by the CBX3-UCOE. Of note, the protective activity of the CBX3-UCOE was associated with decreased levels of repressive histone marks (H3K27me3, H3K9me3) and increased levels of active marks (phosphorylated polymerase). Alternatively, we also targeted an expression cassette to the AAVS1 locus employing zinc finger nuclease mediated homologous recombination. Selected clones showed targeted and robust CMV early enhancer/chicken β actin (CAG) promoter-driven fluorochrome expression in the pluripotent status as well as in terminally differentiated iPSC-MΦ. In summary, we here describe silencing resistance lentiviral vectors and genome editing strategies as well as a differentiation protocol allowing for the efficient and long-term generation of stable genetically modified iPSC-MΦ, thus paving the way for novel gene and cell based therapies.

690. Development of a Clinical Lentiviral Vector for Gene Therapy of SCID-X1

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by profound immunological defects caused by a partial or complete absence of T and NK cells and the presence of non-functional B cells. To overcome the safety issues raised by the use of MLV-based retroviral vectors in previous gene therapy clinical trials, we designed a SIN lentiviral vector (LV) carrying the codon-optimized human IL2RG cDNA under the control of the human EF1αS promoter and a mutated WPRE. Replacement of the native IL2RG open reading frame by a codon-optimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The vector was VSV-G-pseudotyped and produced by a new manufacturing process based on quadri-transfection of suspension-adapted 293T cells grown in serum-free conditions in 50- to 200-L bioreactors, purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The efficacy of this vector was demonstrated in vitro by the restoration of a normal level of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3 and by high efficiency (81±7%) transduction of human mobilized CD34+ hematopoietic stem/progenitor cells with no impact on viability or clonogenic capacity. A biosafety evaluation study of the IL2RG LV in the murine model of the disease showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals. An in vitro assay (IVIM) showed a safe genotoxic profile, while insertion site analysis in transplanted mice revealed a standard lentiviral integration profile and no signs of clonal dominance. These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1.

Chronic granulomatous disease: Clinical, molecular, and therapeutic aspects.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defects in the genes encoding any of the NADPH oxidase components responsible for the respiratory burst of phagocytic leukocytes. CGD is a genetically heterogeneous disease with an X-linked recessive (XR-CGD) form caused by mutations in the CYBB gene encoding the gp91(phox) protein, and an autosomal recessive (AR-CGD) form caused by mutations in the CYBA, NCF1, NCF2, or NCF4 genes encoding p22(phox) , p47(phox) , p67(phox) , and p40(phox) , respectively. Patients suffering from this disease are susceptible to severe life-threatening bacterial and fungal infections and excessive inflammation characterized by granuloma formation in any organ, for instance, the gastrointestinal and genitourinary tract. An early diagnosis of and the prompt treatment for these conditions are crucial for an optimal outcome of affected patients. To prevent infections, CGD patients should receive lifelong antibiotics and antifungal prophylaxis. These two measures, as well as newer more effective antimicrobials, have significantly modified the natural history of CGD, resulting in a remarkable change in overall survival, which is now around 90%, reaching well into adulthood. At present, hematopoietic stem cell transplantation (HSCT) is the only definitive treatment that can cure CGD and reverse organ dysfunction. Timing, donor selection, and conditioning regimens remain the key points of this therapy. In recent years, gene therapy (GT) for XR-CGD has been proposed as an alternative to HSCT for CGD patients without a matched donor. After the failure of the first trials performed with retroviral vectors, some groups have proposed the use of regulated SIN-lentiviral vectors targeting gp91(phox) expression in myeloid cells to increase the safety and efficacy of the GT protocols.

Optimizing CD4+ T-Cells Transduction Protocol for Gene Therapy of HIV-1 Infected Patients

Gene therapy represents an alternative and promising strategy that could provide a path to a curative therapy for HIV-1 infection. One approach involves the introduction of protective gene into a cell, thereby conferring protection against HIV.We plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma. The patients will received autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells. CD34+ and CD4+ will be ex vivo transduced by the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor.We developed a transduction process for CD4+ T-cells using the TransAct™ reagent (Miltenyi Biotec, Bergisch Gladbach , Germany) for CD4+ T-cells activation. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an equivalent efficacy of transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, CD4+ T-cells ex vivo transduced after activation with the TransAct™ reagent display very few changes in their surface marker with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 15-25% of cells suggesting a more "gentle " activation of the transduced CD4+ T-cells.Our transduction process had no significant impact in TCRβ repertoire diversity as evaluated by high-throughput sequencing and analyzis of diversity through the Gini-Simpson index or the Shannon index.Finally, transduced CD4 + T-cells retained the ability to to be primed towards the TH1, TH2 and TH17 pathways suggesting that the transduction protocol used did not alter the functional properties of the target cells. DisclosuresNo relevant conflicts of interest to declare.

Innovative Hematopoietic Gene-Therapy Concepts for Hereditary Pulmonary Alveolar Proteinosis Utilizing Hematopoietic Stem Cell Derived Macrophages

Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce pulmonary transplantation of gene corrected hematopoietic stem cell (HSC)-derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, selective pulmonary engraftment of healthy donor cells upon pulmonary transplantation of MPs was demonstrated by flow- and chip cytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as lung function and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface marker, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following pulmonary transplantation of MPs differentiated from lentivirally corrected Csf2rb -/- HSCs. In vitro these gene-corrected HSCs expanded up to 1045-fold while differentiating into typical alveolar macrophages with F4/80, CD11b, CD11c, CD68, as well as Csf2rb mRNA and protein (CD131) expression and reconstitution of GM-CSF receptor signaling. Transplanted herPAP mice displayed significant improvement of biomarkers in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, administration of human CD34+ cell-derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 human MPs led to long-term pulmonary engraftment and reduced alveolar protein levels by 50-70%. CT scans six months after transplantation revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities and normalisation of inspiratory to expiratory lung volume ratio.Furthermore, to genetically correct human CSF2RA-patient derived CD34+ cells, we have generated SIN-lentiviral vectors expressing a codon-optimized human CSF2RA-cDNA in combination with EGFP (Lv.EFS.CSF2RA.EGFP) from an EFS1a-promoter. BaF3 cells transduced with this vector showed stable and longterm (>3 month) expression of CSF2RA (CD116) by flow cytometry and survived in hGM-CSF dependant assays even at low concentrations of GM-CSF (5 ng/ml) confirming the formation of functional hybrid receptors of the murine GM-CSF receptor ß-chain with the transgene. Characterization of GM-CSF receptor downstream signalling revealed 5-6-fold increased STAT5 phosphorylation by Western blot in response to hGM-CSF (over control cells). Conferring this vector to CD34+ cells of CSF2RA-deficient patients yielded efficient CD116 (CSF2RA) expression, and rescued hGM-CSF dependent colony formation as well as monocytoid differentiation. Of note, clonogenic growth by G-CSF control treatment revealed no differences in colony formation or differentiation capacity when compared to uncorrected patient samples. Furthermore, healthy Lv.EFS.CSF2RA.EGFP transduced CD34+ samples, while showing robust CD116 overexpression, exhibited no aberrations in biological functions such as colony formation or in vitro differentiation towards macrophages.Thus, we here describe an innovative, cause directed and highly effective hematopoietic gene therapy approach to herPAP. Given the longevity of the transplanted MP population in our model, the strategy also may serve as a proof-of-principle to incorporate long-lived differentiated, cell sources into current hematopoietic gene therapy concepts. DisclosuresNo relevant conflicts of interest to declare.

T Cell Products of Defined CD4:CD8 Composition and Prescribed Levels of CD19CAR/Egfrt Transgene Expression Mediate Regression of Acute Lymphoblastic Leukemia in the Setting of Post-Allohsct Relapse

Introduction. ALL relapse following allogeneic HSCT has dismal outcomes due in large part to ineffective therapies. The primary objectives of the Phase 1 portion of this study, which is restricted to the subset of pediatric and young adult patients who relapse following HSCT, is to determine the feasibility of manufacturing products of defined composition and transgene expression, the safety of the cryopreserved T cell product infusion, and to describe the full toxicity profile, including development of clinically significant GVHD. Therapeutic responses are also tracked based on multiparameter flow and IgH deep sequencing.Methods. CD4 and CD8 T cell subsets are immunomagnetically isolated from apheresis products obtained from the research participant. Following anti-CD3xCD28 bead stimulation, T cell lines are transduced with a SIN lentiviral vector that directs the co-expression of the FMC63scFv:IgG4hinge: CD28tm:4-1BB:ζ CAR and the selection/tracking/suicide construct EGFRt. Transduced cells are propagated using recombinant human cytokines to numbers suitable for clinical use over 10-20 days during which time they are subjected to EGFRt immunomagnetic positive selection. Shortly following lymphodepleting chemotherapy, cryopreserved CD4/EGFRt+ and CD8/EGFRt+T cell products are thawed and infused at the bedside such that patients receive a 1:1 ratio of EGFRt+CD4 and CD8 T cells at the protocol prescribed dose level.Results. Six subjects (4m – 3 yr s/p HSCT) have been treated at dose level 1 (5 X 105 CAR T cells/kg); four were treated with active disease, and two were treated while MRD negative. All patients received lymphodepleting chemotherapy prior to T cell infusion. The infusions were well tolerated with only 1 AE > grade 2 (grade 3 anaphylaxis related to the DMSO). All five responding subjects exhibited in vivo expansion of CAR T cells (peak engraftment 13.4 - 93.6 % CAR+ T cells/circulating T cells occurring 8-14 days post infusion) that were predominantly CD8+. Subjects with higher disease burden had higher peak PB CAR T cell levels compared to those with MRD negative marrows (62.7% v 19.6%). Accumulation of CAR T cells in bone marrow and CSF was observed. Five of the six subjects obtained or maintained an MRD negative CR following CAR T cell therapy, while the subject who did not have a therapeutic response failed to have detectable cell product engraftment. Four of the five subjects who had engraftment of CAR T cells have ongoing persistence with accompanying B cell aplasia and leukemia control (35 days-5 months as of August 1, 2014). Only one subject required immunomodulatory treatment for sCRS (tocilizumab and dexamethasone), and that subject lost persistence at day +42. Of the 2 subjects with identified malignant IgH rearrangement, deep sequencing of bone marrow (BM) showed no evidence of the clone by day +21 and day +63, respectively. Each of the five responding subjects developed some degree of CRS with fever and hypotension as the hallmark symptoms (2/6 admitted to ICU). Three of these patients developed encephalopathy (2 grade 1 and 1 grade 4) that was fully reversible. One subject developed de novo grade 2 acute skin GVHD shortly following CAR T cell engraftment and CRS. Skin biopsy revealed that only 9% of skin localized T cells marked EGFRt+ while 79% of circulating T cells marked EGFRt+. This subject was treated with a 2 week course of 1 mg/kg of prednisone, followed by a rapid taper over a six week period and resolution of the GVHD, and has ongoing persistent CAR+T cells.Conclusions. Infusions of 5x105 defined composition CD4:CD8 CD19CAR/EGFRt+ T cells/kg have produced encouraging rates of MRD negative CRs in pediatric and young adults ALL patients who have suffered a postHSCT relapse. Based on intent to treat, we have found it is feasible to generate donor-derived products from each of the six enrolled patients. Expected toxicities include CRS with ~30% ICU admission rate and encephalopathy with severity ranging from mild to severe, but fully reversible. Although one patient developed acute GVHD post T cell therapy, our preliminary assessment suggests that CAR T cells were not mediators of this response. This study continues to accrue at increasing dose levels and updated results will be reported at the meeting. DisclosuresOff Label Use: tociluzimab for treatment of cytokine release syndrome cetuximab for the ablation of T cells CAR T cells for the treatment of leukemia. Jensen:Juno: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Generation of SIN-Lentiviral Vectors for the Treatment of Canavan Disease Using Ex Vivo Transduction of Hematopoietic Target Cells

The autosomal recessive leukodystrophy Canavan Disease is caused by an acquired or hereditary mutation in the Aspartoacylase gene (ASPA) leading to enzymatic dysfunction of a critical component of central nervous system metabolic and osmotic homeostasis. This loss of enzyme function results in demyelination, hydrocephalus, spastic paralysis, seizures, and ultimately premature death. Metabolic treatment for Canavan Disease can slow but not stop disease progression. Gene therapy aimed at replacing the defective ASPA gene has been employed in 13 patients using AAV-2 vectors expressing the human wild-type ASPA cDNA driven by a human Neuron Specific Enolase promoter and delivered via intra-parenchymal injection. Long-term follow-up in these patients revealed minimal clinical impact as although there was measurable physiologic activity of the transgene, it was likely less than what would be sufficient to alter the disease. It has been clearly demonstrated in animal models and in human patients that vector transduced hematopoietic long-term repopulating cells can differentiate into self-renewing microglial populations that can seed the CNS following busulfan treatment. To improve gene delivery to the CNS we have chosen to target the hematopoietic system with SIN-lentiviral vectors expressing wild-type ASPA linked via picornavirus 2A sequence to a GFP reporter gene driven by the highly active MND promoter and flanked by the 650bp cHS4 insulator element. We have produced this vector at laboratory scale with unconcentrated titers in the low 107 t.u./ml range. We have demonstrated transmission of full length vector genome to both cell lines and human CD34+ primary cells. Single cell jurkat transducents were expanded to clonal cell lines which all demonstrated measurable ASPA protein by Western blot and functional ASPA enzyme activity by HPLC assay. Transduction of human mobilized peripheral blood CD34+ cells at an MOI of 26 (MOI 13 x 2 exposures) and plated in methylcellulose for CFU-C assay resulted in 40.6% GFP+ cells by flow cytometry and 0.69 vector genomes/ human genome by real-time PCR. Vector and mock treated cells were transplanted into busulfan conditioned NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and allowed to engraft. After 12 weeks in vivo, mice were sacrificed and the bone marrow extracted. No difference in overall (hCD45+) or lineage specific (hCD33+, 19+, or 71+) human cell engraftment was observed between the arms indicating a lack of toxicity, with a transduction efficiency of 15.3±7.4% GFP+ hCD45+ cells by flow cytometry and 0.23±0.15 vector genomes/ human genome by real- time PCR. Based on the success of these proof-of-concept experiments, we have produced reporterless versions expressing wild-type and human codon-optimized ASPA. We plan to test these vectors for hematopoietic toxicity and transduction efficiency in human primary cells using the NSG mouse xenotransplant model and to test for functional correction in the Nur7 Canavan Disease mouse model by transducing 5-FU treated Nur7 mouse bone marrow cells and transplanting them into busulfan conditioned Nur7 recipients. If these vectors continue to perform well in in vivo preclinical assays, it is our plan to begin large-scale production with translation into clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Lentiviral MGMTP140K-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter

Notwithstanding recent successes, insertional mutagenesis as well as silencing and variegation of transgene expression still represent considerable obstacles to hematopoietic gene therapy. This also applies to O6-methylguanine DNA methyltransferase (MGMT)-mediated myeloprotection, a concept recently proven clinically effective in the context of glioblastoma therapy. To improve on this situation we here evaluate a SIN-lentiviral vector expressing the MGMTP140K-cDNA from a combined A2UCOE/PGK-promoter. In a murine in vivo chemoselection model the A2UCOE.PGK.MGMT construct allowed for significant myeloprotection as well as robust and stable selection of transgenic hematopoietic cells. In contrast, only transient enrichment and severe myelotoxicity was observed for a PGK.MGMT control vector. Selection of A2UCOE.PGK.MGMT-transduced myeloid and lymphoid mature and progenitor cells was demonstrated in the peripheral blood, bone marrow, spleen, and thymus. Unlike the PGK and SFFV promoters used as controls, the A2UCOE.PGK promoter allowed for sustained vector copy number-related transgene expression throughout the experiment indicating an increased resistance to silencing, which was further confirmed by CpG methylation studies of the PGK promoter. Thus, our data support a potential role of the A2UCOE.PGK.MGMT-vector in future MGMT-based myeloprotection and chemoselection strategies, and underlines the suitability of the A2UCOE element to stabilize lentiviral transgene expression in hematopoietic gene therapy.