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Chronic granulomatous disease (CGD) is an inherited blood disorder of phagocytic cells that renders patients susceptible to infections and inflammation. A recent clinical trial of lentiviral gene therapy for the most frequent form of CGD, X-linked, has demonstrated stable correction over time, with no adverse events related to the gene therapy procedure. We have recently developed a parallel lentiviral vector for p47phox-deficient CGD (p47phoxCGD), the second most common form of this disease. Using this vector, we have observed biochemical correction of CGD in a mouse model of the disease. In preparation for clinical trial approval, we have performed standardized preclinical studies following Good Laboratory Practice (GLP) principles, to assess the safety of the gene therapy procedure. We report no evidence of adverse events, including mutagenesis and tumorigenesis, in human hematopoietic stem cells transduced with the lentiviral vector. Biodistribution studies of transduced human CD34+ cells indicate that the homing properties or engraftment ability of the stem cells is not negatively affected. CD34+ cells derived from a p47phoxCGD patient were subjected to an optimized transduction protocol and transplanted into immunocompromised mice. After the procedure, patient-derived neutrophils resumed their function, suggesting that gene correction was successful. These studies pave the way to a first-in-man clinical trial of lentiviral gene therapy for the treatment of p47phoxCGD.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy., In recent years, hematopoietic stem cells gene editing has emerged as a promising tool to treat blood disorders. Here the authors develop a CRISPR/Cas9-based genome editing strategy that allows the precise correction of Wiskott-Aldrich Syndrome in vitro and in vivo with high efficiency.

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (ALL)1–5, but toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, limits broader application. Moreover, 40–60% of patients relapse owing to poor CAR T cell persistence or emergence of CD19− clones. Some factors, including the choice of single-chain spacer6 and extracellular7 and costimulatory domains8, have a profound effect on CAR T cell function and persistence. However, little is known about the impact of CAR binding affinity. There is evidence of a ceiling above which increased immunoreceptor affinity may adversely affect T cell responses9–11. We generated a novel CD19 CAR (CAT) with a lower affinity than FMC63, the high-affinity binder used in many clinical studies1–4. CAT CAR T cells showed increased proliferation and cytotoxicity in vitro and had enhanced proliferative and in vivo antitumor activity compared with FMC63 CAR T cells. In a clinical study (CARPALL, NCT02443831 ), 12/14 patients with relapsed/refractory pediatric B cell acute lymphoblastic leukemia treated with CAT CAR T cells achieved molecular remission. Persistence was demonstrated in 11 of 14 patients at last follow-up, with enhanced CAR T cell expansion compared with published data. Toxicity was low, with no severe CRS. One-year overall and event-free survival were 63% and 46%, respectively. New low-affinity anti-CD19 CAR T cells exhibit peripheral expansion and persistence without inducing severe cytokine-response syndrome in pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (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. Disclosures Ghorashian: 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.

Relapsed and refractory B-lineage acute lymphoblastic leukemia remain the leading cause of cancer related death in children and young adults. Clinical studies of adoptive cell immunotherapy, re-directing T cells against CD19 by endowing them with a chimeric antigen receptor (CAR), have shown considerable clinical responses. To date, 3 different binding domains (scFv) targeting CD19 have been used in CARs taken forward in clinical trials and we have constructed a new CD19-CAR, derived from a different anti-human CD19 antibody, clone CAT. Whether different binding affinities of the CD19 targeting domain, when significantly different, could affect CAR-mediated T cell functionality has not been evaluated in depth. We therefore investigated the impact of scFv affinity on CAR-mediated T cell function in vitro, as well as on anti-tumour efficacy in vivo. We have generated 3 CD19-CARs only differing in their scFv, which were derived from 3 anti-human CD19 antibodies (Clones FMC63, 4G7 & CAT) respectively. All other structural variables of the CAR and the use of the 4-1BB endodomain were identical. The Kd values obtained by Biacore Surface Plasmon resonance (SPR) analysis ranged from 8.8 x 10-10 to 1.1 x 10-7. Differences in affinity were predominantly determined by the off-rates, leading to significantly quicker dissociation from its target in CAT scFv compared to FMC63 and 4G7. CAT-CAR transduced T-cells showed enhanced cytotoxic responses to the CD19+ cell line SUPT1-CD19 in 51Cr release assays (p A quick dissociation rate has been described to be of particular importance when targeting cells with low levels of antigen expression, as T cell functional avidity can be detrimentally affected when dissociation is prolonged (Thomas et al, Blood 2011). We therefore investigated cytotoxicity of CAR transduced T cells against a cell line engineered to express CD19 at very low levels. This demonstrated increased cytotoxicity by CAT+ T-cells as well as greater CD107a degranulation in response to low CD19 expressing targets compared to FMC63 or 4G7-transduced T cells. Similarly, CAT+ T-cells showed greater killing of NALM 6 cells at very low effector:target ratios, reflecting the ability of serial killing by CAT+ T-cells by virtue of their rapid dissociation from target cells. Live cell imaging studies by confocal microscopy analysis confirmed a higher number of serial engagements by CAT+ T-cells (p We are now studying the relative potency in a xenogeneic model of ALL, using a CAR-T cell dose that is purposefully lowered to a suboptimal range to study kinetic differences and tumor clearance. Preliminary data suggests that, transferred after exposure to leukemia, CAT+ T cells have a less exhausted phenotype and higher effector:target ratios 2 weeks after infusion. Further experiments, in which recipient mice are re-challenged with the same tumor, will assess differences in the ability of adoptively-transferred CAR T cells to form memory. In conclusion, we have developed a novel CD19-CAR which confers enhanced cytotoxicity and proliferative responses compared to existing CD19-CARs. Our work indicates that the scFv binding kinetics impacts the functional avidity of CAR-transduced T cells, providing important implications for the design of future CARs, especially when tumour cells expressing low levels of antigen are targeted. Disclosures Onuoha: Autolus Ltd: Employment, Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

It is now widely accepted that in chronic lymphocytic leukaemia (CLL), proliferation of the malignant B cell clone takes place in pseudofollicles positioned within lymphoid tissue. This complex environment is believed to provide CLL cells with signals necessary for survival and expansion. In recent years the role for T cells in these processes has started to emerge. By analysing formalin fixed paraffin embedded (FFPE) CLL lymph node (LN) sections by multi-parameter confocal microscopy, our group previously demonstrated that dividing CLL cells stay in close contact with CD4+ CD25+ Foxp3- T cells. In addition, in an in vitro assay, activated T cells were found to be capable of inducing CLL cell proliferation. Our subsequent flow cytometry analyses looked at the phenotype of the T cells isolated from the CLL lymph nodes by fine needle aspiration (FNA). LN T cells were found to express effector memory cell markers more commonly than their PB counterparts and also had much higher levels of the activation/exhaustion marker PD1. Since PD1 is known to be a marker of T follicular helper cells (Tfh), in our current work we asked if Tfh cells are a significant component of the T cell infiltrate of CLL lymph nodes. We used confocal immunofluorescent microscopy on FFPE CLL LN sections and looked for coexpression of CD4, PD1 and ICOS – also used to identify Tfh cells. PD1 was expressed on 25% of CD4+ cells (95% CI 21-30) but only 4% of CD4+ cells expressed both PD1 and ICOS (95% CI 2.5-5.6) (n=6). The number of Tfh cells infiltrating CLL LN was shown to be low when compared with the numbers found in normal reactive LN germinal centres (33.3% ±3.0 CD4+ cells co-express PD1 and ICOS) and more closely resembled the interfollicular areas of the normal reactive LN, where 0.71% ±0.23 CD4+ cells co-express PD1 and ICOS (n=6). As PD1 is expressed on chronically activated T cells, we next sought evidence for CLL T cell antigen-specificity. LN-FNA and matching peripheral blood (PB) CD4+ T cells from 6 CLL patients were sorted into PD1hi and PD1lo subsets and subject to spectratyping of their T cell receptor Vβ (TCRVβ) repertoire. Diversity was then assessed using an arbitrary scale in which a higher value indicated a loss of TCR diversity. There was a significant reduction in TCR diversity observed in the PD1hi subset in both LN and PB compartments compared to PD1lo T-cells (PB PD1hi vs PD1lo; 19.83 +/- 1.62 vs 14.67 +/- 2.42; p=0.0049; LN PD1hi vs PD1lo - 21.33+/-0.56 vs 16.00+/- 1.77; p=0.022). Importantly, CLL PB PD1hi cells were also shown to have a significantly reduced TCR diversity compared to PB PD1hi cells from normal age-matched controls (19.83+/-1.62 vs 12.67+/-2.54; p=0.039; n=6). TCR oligoclonality observed in the CLL LN and PB PD1hi CD4 T cells strongly supports the role of antigen in maintaining these two populations. Since PD1hi cells were far more frequent in the CLL LN than blood, these antigen driven interactions likely occur within the LN. In order to look for further evidence of CLL LN T cell antigen-specificity we then performed high throughput TCRVβ CDR3 sequencing (Illumina Genome Analyser, analysed by Immunoseq; Adaptive Biotechnologies) of CLL LN-FNA and PB CD4+ cells. Analysis of TCR sequences obtained from two separate LN and matching PB samples sampled concurrently from the same individual (n=4 patients) revealed that there is a significantly higher commonality in TCR CDR3 clonotypes between two matching LN samples then between any LN and its matching PB sample (p=0.016). These results support the theory that the CLL LN is a site of specific antigen stimulation of CD4+ T cells. Further support for this hypothesis came from the analysis of TCRVβ sequencing results from two sets of two CLL LN and PB samples collected from the same individual 1 month apart. Six persistent TCR CDR3 clonotypes were detected, which were present in all four LN but not in the PB. These clones made up around 1% of the total number of TCR sequences found in each of the LN. In conclusion, our data provides strong new evidence that, just like the neoplastic B cells, CLL LN CD4+ T cells are antigen experienced and that their accumulation in CLL lymph nodes may be antigen-driven. Disclosures: No relevant conflicts of interest to declare.