Term Engraftment Research Articles

Bispecific CAR-iNKT Immunotherapy for High Risk MLL-Rearranged Acute Lymphoblastic Leukemia

MLL-rearranged CD19+ acute lymphoblastic leukemia ( MLLr-ALL), the commonest infant leukemia, has a high mortality rate (<40% leukemia-free survival). While stem cell transplantation has not improved outcomes, CD19-directed immunotherapeutic approaches, including CAR-T cells, have shown promise. However, as patients can still relapse with CD19-, or lineage-switched disease, alternative strategies are needed. To enhance efficacy and mitigate against immune escape, we aimed to develop iNKT cell-based bispecific CAR immunotherapy. iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. Furthermore, allogeneic iNKT protect from aGVHD making them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. To target both CD19 and CD133, which is often co-expressed with CD19 on MLLr blasts and possibly also on CD19-CD133+ leukemia-initiating cells, we developed a CD19-28z/CD133-41BBz bispecific as well as CD19-28z and CD133-4-1BBz monospecific CARs and compared their anti-leukemic activity. In short- and long-term cytotoxicity assays against MLLr leukemic cell lines (SEM, RS4;11, KOPN8) we show that bispecific CAR-iNKT are more potent than their monospecific counterparts. In assays involving single or dual knock-out of CD19 and CD133 in SEM cells, both the CD19 and CD133 moieties of the bispecific CAR were reactive and specific against their respective targets. While bispecific CAR-iNKT retained killing activity against either single target-expressing leukemia cells, monospecific CAR-iNKT only killed leukemia cells expressing their cognate target. In line with the in vitro findings, in xenograft assays, a low dose of 10 6 bispecific CAR-iNKT cells significantly prolonged survival of luc-SEM leukemia-bearing mice as compared to monospecific CAR-iNKT-treated and untreated control animals (Fig 1A). Of note, in vivo as well as in vitro, bispecific CAR-iNKT very effectively eliminated high and low CD19/CD133 co-expressing, but not, as expected, CD19-CD133- disease. Next, we compared bispecific CAR-iNKT vs their CAR-T counterparts. In vitro, CAR-iNKT from three donors demonstrated higher killing activity than CAR-T, while in vivo, compared to 10 6 CAR-T, CAR-iNKT cells induced significantly improved survival of leukemia-bearing mice (Fig 1B). Of note, while CAR-T cells were not detectable in the BM and spleen at sacrifice, CAR-iNKT were readily detectable in the organs of CAR-iNKT cell-treated animals, suggesting they persist longer. Remarkably, mice treated with 5x10 6 bispecific CAR-iNKT cells survived >60 days without any evidence of leukemia by serial bioluminescence assay, or in the bone marrow, spleen (flow-cytometry) and leptomeninges (immunohistochemistry) upon culling. Since SEM cells do not express CD1d, we explored alternative mechanism(s) that would account for the more potent anti-leukemic activity of CAR-iNKT cells. We found that CAR-iNKT express significantly higher baseline levels of the activating receptor NKG2D than CAR-T cells. Upon co-culture with SEM cells, nearly all CAR-iNKT but only a fraction of CAR-T express NKG2D, and a blocking NKG2D Ab significantly abrogated the superior cytotoxic effect of CAR-iNKT cells. Consistent with an NKG2D-dependent mechanism, mRNA of one or more NKG2D ligands is expressed by primary MLLr blasts as well as leukemia cell lines. Since CD133 is expressed on hematopoietic stem and progenitor cells (HSPC), the potential hematopoietic toxicity of bispecific CAR-iNKT cells was tested in a series of experiments involving cord blood humanisation of NSGS mouse hematopoiesis. We found that transfer of 10 7 bispecific CAR-iNKT cells significantly but transiently depleted engrafted CB CD19+ B cells. However, phenotypic analysis showed that long term engraftment levels were similar for all mature cell lineages, CD34+ HSPC and specifically for Lin-CD34+CD38- HSC. We conclude that bispecific CAR-iNKT cell immunotherapy is a very effective treatment for pre-clinical aggressive MLLr-ALL, it outperforms CAR-T cell immunotherapy in an NKG2D-dependent manner and has the potential to protect from immune escape, leptomeningeal disease and lineage switch without discernible hematological toxicity. These findings provide the basis for clinical development of bispecific CD19/CD133 CAR-iNKT cells as an ‘off-the-shelf’ treatment for MLLr-ALL.

Highly Efficient & Specific Repair of MAGT1 Mutation in Xmen Patient T Cells and Hematopoietic Stem Cells

Introduction: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) is a congenital glycosylation disorder with combined immune deficiency due to Magnesium Transporter 1 (MAGT1) mutations. Defective glycosylation of Natural Killer Group 2D (NKG2D), an activating receptor expressed on CD8 and NK cells critical for cytotoxicity, results in EBV lymphoproliferative disease and an inverted CD4:CD8 ratio in many XMEN patients. Oral magnesium supplementation for XMEN patients was not helpful, and hematopoietic stem cell transplants (HSCT) are complicated by high mortality. CRISPR-Cas9-AAV inserts MAGT1 specifically at endogenous locus but relies on double strand DNA breaks and adeno-associated virus donor that triggers DNA damage response. To address this, we evaluated base editing approach to repair a MAGT1 mutation that affects multiple patients with XMEN disease. We observed highly efficient mutation repair of XMEN T cells (75.5%) and hematopoietic stem cells (71.9%) to restore MAGT1 function and persistence of gene correction following transplant into immunodeficient mice. Materials and methods: Peripheral blood hematopoietic stem cells (HSPCs, CD34+) and mononuclear cells (PBMC) were collected from XMEN patients and healthy donors (HD) after G-CSF and plerixafor mobilization. PBMCs were stimulated (anti-CD3/anti-CD28, IL-2 (100 IU/mL), in ImmunoCult™-XF T cell expansion medium for 5-7 days prior to editing. HSPCs were cultured in StemSpan™ SFEM II Stem Cell Factor, Thrombopoietin, Fms-like tyrosine kinase 3 ligand (all 100ng/mL) and UM171 for two days prior to editing. Base editing is performed by electroporation (MaxCyte Inc.) of cells with ABE8e SpRY (CELLSCRIPT LLC.) and synthetic guide (Synthego) RNAs. For in vitro killing activity, T cells were co-cultured with EBV-B cells at a ratio of 1:1 for 48 hours and analyzed for apoptosis (Apoptracker Green+ 7AAD-), necrosis (Apoptracker Green+ 7AAD+) and degranulation (CD8+ CD107a+). HSPCs were transplanted into mice two days after EP and harvested 17-20 weeks after transplant. Results:Base editing of XMEN T cells: Flow cytometric analysis 2 days post BE show restored expression of biomarker NKG2D in XMEN CD8+ T cells (75.5%) compared to HD (93.2%) and naïve XMEN (2.39%). CD8+ cytotoxic activity against EBV-B cells was restored in BE XMEN patient cells; as indicated by increased apoptosis (BE: 6.78%, naïve: 4.82%, HD: 8.17%), necrosis (BE: 9.35%, naïve: 4.54%, HD: 7.19%), and degranulation (BE: 22.80%, naïve: 15.50%, HD: 21.40%), compared to naïve MAGT1 CD8+ T cells.In an in vivo tumor model, BE XMEN T cells injected into mice with induced luminescent EBV-B cell tumors lysed tumors and reduced average bioluminescence flux (photons/sec) (7 weeks) (8.23 × 107 ± 1.06 × 108) compared to naïve T cells (3.39 × 109 ± 2.22 × 109 and HD T cells (1.45 × 109 ± 2.22 × 109). Base editing of HSPCs: Immunodeficient NSGS pups transplanted with BE XMEN HSPCs engrafted and differentiated normally into lymphocytes with a normalized CD4:CD8 ratio in spleen (BE: 0.79 ± 0.14, naïve: 3.45 ± 1.1, HD: 1.3 ± 0.40) and peripheral blood (BE: 2.16 ± 0.84, naïve: 5.56 ± 2.32, HD: 1.79 ± 0.70) (Fig 1a). Importantly, restoration of biomarker NKG2D expression selectively on CD8+ T cells in spleen (BE: 80.5% ± 8.61%, naïve: 3.63% ± 1.45%, HD: 68.9% ± 10.32%) and peripheral blood (BE: 86.1% ± 2.88%, naïve: 2.25% ± 1.45%, HD: 62.6% ± 12.44%) (Fig 1b) confirmed correction of MAGT1 function in the glycosylation of critical NKG2D protein. High throughput targeted sequencing of on-target locus shows minimal bystander edits and additional off-target analysis is ongoing. Conclusion: Our data show highly efficient and specific base editing repair of MAGT1 mutation in XMEN T lymphocytes and CD34+ HSPCs. BE XMEN T cells increased cytotoxic activity in vitro and killing of tumor cells in mice. In vivo studies with mice transplanted with BE CD34+ HSPCs also confirm long term engraftment and differentiation into CD8+ T cells with restored NKG2D necessary for activation and cytolytic activity of CD8+ T and NK cells. The highly efficient and specific correction with base editing holds great promise for gene therapy for XMEN disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single Cell Analysis of Adult Human Hematopoietic Stem and Progenitor Cells Identifies a Novel Lymphoid Primed Multipotent Progenitor That Expands in Relapsed Acute Myeloid Leukemia

Background: Hematopoietic stem and progenitor cells (HSPCs) are capable of generating all human blood cells. Although these cells have been extensively evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al. Nat. Commun. 2019), there remains significant uncertainty as to the degree of heterogeneity within HSPC subpopulations and their relevance to disease. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of numerous blood disorders including hematologic malignancies. To address this need, we extensively characterized healthy bone marrow mononuclear cells (BMMCs) with both whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and multiplexed (n=35) surface marker analysis, we could not only improve HSPC clustering but also characterize specific phenotypic states along unique hematopoietic differentiation trajectories. This analysis allowed us to identify new subpopulations within currently defined hematopoietic stem cells (HSCs), multipotent progenitors (MPPs) and lymphoid primed multipotent progenitors (LMPPs).Methods: Three healthy adult BMMC samples were stained with antibody conjugated oligonucleotides (BD Abseq) and analyzed using a nanowell cell capture system (BD Rhapsody). We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. These cells were first clustered using matrix factorization in order to identify unique HSPC states. Novel clusters within the HSC, MPP, and LMPP subpopulations were purified using flow cytometry, and functionally evaluated with both in vitro and in vivo assays.Results: The antibody-derived tags (ADTs) obtained from BD Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 versus 934 for mRNA alone for 20 clusters). Additionally, we were able to design a targeted HSPC panel (n=500 genes) with Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (Rand index = 0.88). HSPC clustering identified the putative hematopoietic stem cell (HSC), a multipotent progenitor (MPP), and a new lymphoid primed multipotent progenitor (LMPP). A new computational sorting strategy was derived to purify these primitive HSPCs, and subsequently validated with flow cytometry. The functional evaluation of these sorted populations revealed that HSC and MPP cells were capable of increased serial replating ability in vitro suggesting enhanced self-renewal capabilities compared to LMPP cells. All three HSPC subpopulations produced gradients of erythroid and myeloid colonies in methylcellulose, and T-cells in T-cell expansion assays in vitro. The HSCs were the only cells that were able to produce long term engraftment in immunodeficient mice after serial transplantations. Additionally, common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers were identified. The primitive HSPC clusters were converted into a signature matrix using CIBERSORTx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. Using multivariate Cox proportional hazard analysis, we found that high levels of MPPs at diagnosis was the strongest feature associated with worse overall survival (HR = 78.46, 95% confidence interval 7.32-828). Interestingly, after performing paired analysis of diagnostic and relapse cases, the HSC and LMPP expanded significantly in relapsed AML after chemotherapy (p < 0.05) whereas the MPP expanded considerably after stem cell transplant but did not reach significance (p = 0.09).Conclusions: In summary, we identified new cell type clusters within previously defined HSC, MPP, and LMPP subpopulations, and unique surface marker associations using combined single cell WTA and surface marker analysis. We were able to purify these primitive HSPCs using these new markers, in addition to classical markers like CD34, CD90, and CD45RA. Importantly, deconvolution analysis provided preliminary insights into their clinical relevance in AML. DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc: Membership on an entity's Board of Directors or advisory committees; Circbio inc: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutics Solutions Inc: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead Sciences, Inc: Patents & Royalties.

Mass Cytometry Reveals the Broad Effect of CMV Reactivation on Adaptive and Innate Immune Recovery in HSCT Patients Undergoing Adoptive T Cell Therapy for Viral Infections

Introduction. Adoptive cell therapy (ACT) with virus specific T cells (VST) has been shown to be safe and effective in controlling viraemia in recipients of haemopoietic stem cell transplant (HSCT). Long term engraftment has been demonstrated in some studies but the fate of the transferred cells is not well understood. We hypothesised that the clinical effect of ACT is mediated by interaction of transferred cells with a wide range of innate and adaptive immune cells present in the recipient at the time of ACT treatment. In order to better understand the interaction of stem cell donor-derived and adoptively transferred third-party cells, we sought to apply immune profiling by mass cytometry.Methods. Longitudinal samples from healthy individuals and collected pre- and post-donor derived and third party ACT in HSCT recipients were assessed with a 37 marker panel to identify canonical immune subsets. Cryopreserved peripheral blood mononuclear cells were thawed in batches and stained with metal-labelled antibodies and markers for DNA content and viability. Samples were acquired on the Helios upgraded CyTOF2 mass cytometer. Following sample QC and normalisation of files, viable cells were manually gated to 75 canonical populations with cell subsets enumerated and represented graphically for each sample using visualised t-stochastic neighbour embedding (ViSNE). Cell subsets were expressed as percent of total cells as well as absolute cell number (x109/L) using lymphocyte and monocyte counts from an automated whole blood analyser. This approach was taken to incorporate the large numerical changes that occur in the early months post-HSCT. Mass cytometry data was analysed with unsupervised and supervised clustering algorithms to identify immune signatures. Significance of microarray (SAM) was used to identify the cell subset changes that contributed to the differences in clusters. t-stochastic neighbour embedding (tSNE) and principal component analysis (PCA) were used to correlate immune signatures with clinical variables including patient, transplant and donor baseline characteristics and post-transplant outcomes (including CMV reactivation and disease, acute GVHD, relapse and death).Results. A total of 104 samples from 9 healthy donors and 27 HSCT recipients who received ACT on phase I and II clinical trials were available for analysis. ViSNE revealed highly detailed relationships of the 75 cell subsets within individuals longitudinally and between patients. Unsupervised clustering on all patients and time points and the healthy individuals revealed three distinct immunological signatures (Figure 1). Healthy individuals (signature A) were clearly distinguished from all patients and no patient developed a normal immune profile over the timeframe of the study (up to 6 months post HSCT). In a subset of samples from HSCT recipients a distinct immune phenotype was seen (signature C), characterised by increased CD8+ subsets (CD8em, CD8emra (both of these with and without PD1), CD8+CD57+, CD8+CD31+), NK cell subsets (CD16-, NKp46loCD56lo), NKT cells and plasmacytoid dendritic cells. Of the clinical parameters assessed, signature C was most strongly correlated with CMV reactivation. Signature B showed lower cell numbers overall and included patients with acute GVHD treated with corticosteroids and patients in whom CMV reactivation progressed to CMV disease.Conclusion. Mass cytometry and sophisticated analysis reveal an unprecedented level of detail about the relationships of immune subsets to one another at single time points and their changes over time. This approach identifies distinct global immune signatures involving changes to multiple immune subsets in patients undergoing HSCT and ACT. We have identified an immune signature associated with CMV reactivation that indicates a broader effect of CMV reactivation on immune recovery than previously recognised. As this study was not randomised and did not include a control group who were not treated with ACT, further investigation is required to define the contribution of ACT to the observed immunological changes. This global approach to immunological assessment has the potential to improve understanding of the dynamics of immunological recovery post HSCT. [Display omitted] DisclosuresGottlieb:Indee: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Wave-Complex-Dependent Actin Dynamics Controls Hematopoietic Stem Cell Bone Marrow Retention and Orderly Differentiation into Mature Hematopoietic Lineages

BackgroundSpatiotemporally controlled actin dynamics are crucial for a vast number of cellular functions including migration, proliferation, attachment or immunological synapse formation. Environmental signals activate cognate surface receptors which in turn engage specific small GTPases. Signal integrators (e.g. WASP, WAVE complex or WIP) propagate the signal to Arp2/3 complex and lead to immediate actin polymerization. Here we show that disruption of one of the actin polymerization pathways through specific targeting of WAVE complex disturbs hematopoietic stem and progenitor cells (HSPC) bone marrow retention and their sequential differentiation towards mature blood cells.MethodsWe made use of the recently described mouse model of Hem1 ablation. Hem1 is a strictly hematopoietic specific component of WAVE complex; its loss disrupts the entire WAVE complex exclusively in cells of hematopoietic origin. An array of in vitro assays was employed to test cell intrinsic effects of Hem1 ablation on actin polymerization dependent cellular functions. In vivo transplantation experiments were performed to study the impact of Hem1 ablation on immature and in consequence on mature hematopoiesis.ResultsHem1-/- HSPC display a bone marrow (BM) retention defect evidenced by strongly elevated numbers of circulating HSPC and 50% reduced BM cellularity. In line with that, migration towards CXCL12, a key BM anchor for HSPC, was completely abrogated in Hem1-/- HSPC. Attachment to feeder cells was also decreased by 35% compared to WT HSPC, although stress fiber formation upon CXCL12 stimulation or general ability to polarize was not affected. Next we tested whether this cellular phenotype had any impact on HSPC performance in vivo. Homing, the initial interaction of transplanted HSPC with BM environment, was not affected by Hem1 ablation, while engraftment, the ability to repopulate all mature hematopoietic lineages, was strongly diminished. Platelet and RBC engraftment was delayed, whereas Hem1-/- neutrophils did not engraft at all (Figure 1). Hem1-/- mice presented with chronic inflammation, hyperleukocytosis and elevated pro-inflammatory cytokine plasma levels. To rule out inflammation as causal for numeric and functional Hem1-/- HSPC depletion we performed co-transplantation experiments. Hem1-/- or WT BM was transplanted in a four-fold excess over GFP+ competitor BM. In Hem1-/- transplanted recipients, already 4 weeks after transplantation 60 to 90% of both myeloid and lymphoid compartments were GFP+, whereas, as expected, in the control experiment WT BM out-competed GFP+ BM over time. A similar picture emerged in secondary recipients. Again Hem1-/- BM completely lost against, whereas WT BM prevailed over GFP+ competitor. We furthermore confirmed that the Hem1-/- HSPC cell intrinsic defect is already apparent during embryonic development: As in BM of adult Hem1-/- mice, HSPC content in E14.5 old fetal livers (FL) was reduced by half compared to WT littermates. Also the competitiveness of FL HSPC was strongly impaired, similar to Hem1-/- adult BM HSPC. Whereas short term engraftment was provided by more mature competitor GFP+ BM in both recipients groups, already 4 weeks after transplantation, as expected, WT FL derived HSPC took over blood cell production and completely outcompeted GFP+ BM. In Hem1-/- FL recipients, by contrast, the contribution of competitor GFP+ BM to hematopoiesis remained high for the whole period tested without significant contribution of Hem1-/- FL hematopoiesis (Figure 2). Taken together, Hem1 (and, by extension, WAVE complex) governs HSPC BM retention and proper engraftment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Frequency of Both, Short and Long-Term Post-Transfusion Microchimerism Among the Regularly-Transfused Thalassemic Patient Population

Background: Transfusion of blood products has been associated with the long term engraftment of donor cell-derived genetic material in the recipient (microchimerism: MC), whose clinical implications have not been sufficiently studied. The manifestation of post-transfusion MC has mainly been studied among severe trauma injured patients, heavily transfused at short term periods of time.Aim: We investigated the presence of post-transfusion MC among the regularly-transfused thalassemic patient population of Western Greece, and we analyzed factors, which might influence the frequency and severity of this phenomenon.Patients - Methods: Sixty-four transfusion-dependent homozygous β-thalassemic patients [female (N=32), male (N=32), with a median age of 42 years (range 22-60 years), completely transfusion-dependent (N=54) or partially-dependent (n=10) and 21 never-transfused male controls (median age 41 years, range 24-53 years) were studied. To reveal the presence of MC we have used a real-time PCR technique with SYBR Green, for the detection of 12 HLA-DR alleles. To enhance the ascertainment of MC we used primers corresponding to 12 loci among eight somatic chromosomes that have relatively common, phenotypically silent biallelic insertion or deletion polymorphisms (SO-1 through SO-11). All reactions were performed in double and positive samples were re-tested in double. Overall, 205 samples were tested, and samples from 57 patients were re-tested, after a median of 9.5 months. Moreover, a group of 21 patients, randomly selected, was tested for a third time, after a median of 17.6 months from the second test and 26.4 months from the baseline test.Results: Fifty-one patients (79.7%) and 8 controls (38%) exhibited a positive reaction to 1-7 chimeric alleles (patients) and 1 or 2 alleles (controls). One patient could not identified. Twenty patients (31.2%) and 6 controls (28.5%) have shown positivity for 1 chimeric alleles. Sixteen patients (25%) and 2 controls (9.5%) have shown positivity for 2 chimeric alleles. All the other positive patients but no control subject, had a positive signal for 3 or more alleles. Overall, 21 patients had evidence of long-term MC, and 8 patients only short-term MC, whereas 22 patients detected with both, short- and long-term MC. The detection of positivity in the control subjects could be interpreted as persistence of feto-maternal MC. As expected, there was a female predominance in MC detection, with higher frequency of positive samples (30/32 ~ 93.7% vs 22/32 ~ 68.7%, p<0.01) and higher mean number of positive alleles per subject, compared to males (x±SD: 2.46±1.69 vs 1.15±0.97, p=0.0004). Patients younger than 44 years had lower number of positive signals (1.06±1.5 vs 2.21±1.89, p=0.06), and the same was also true for patients with lower transfusion frequency (<6 units per year: 0.90±0.83), compared to moderately (6-20 units per year) or intensely transfused patients (>20 units per year: 2.0±1.63, p=0.045). Moreover, splenectomized patients had also higher mean number of chimeric alleles, compared to non-splenectomized (2.13±1.76 vs 1.39±1.01, p=0.054). Finally, in 7 of the 21 patients, which MC persisted at all 3 time points, we detected the gradual establishment of 6 new chimeric polymorphisms, but also the disappearance of 4 short-term chimeric polymorphisms, during a median period of 26.4 months.Conclusion: High frequency of regularly transfused thalassemic patients establish long-term microchimerism, which is an active and evolving phenomenon, related to transfusion burden and is influenced by patient's gender, age and splenectomy. The clinical impact of this phenomenon is as yet unknown and unpredicted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Dose Versus Low Dose Syngeneic Hepatocyte Transplantation in Pex1-G844D NMRI Mouse Model is Safe but Does Not Achieve Long Term Engraftment.

Genetic alterations in PEX genes lead to peroxisome biogenesis disorder. In humans, they are associated with Zellweger spectrum disorders (ZSD). No validated treatment has been shown to modify the dismal natural history of ZSD. Liver transplantation (LT) improved clinical and biochemical outcomes in mild ZSD patients. Hepatocyte transplantation (HT), developed to overcome LT limitations, was performed in a mild ZSD 4-year-old child with encouraging short-term results. Here, we evaluated low dose (12.5 million hepatocytes/kg) and high dose (50 million hepatocytes/kg) syngeneic male HT via intrasplenic infusion in the Pex1-G844D NMRI mouse model which recapitulates a mild ZSD phenotype. HT was feasible and safe in growth retarded ZSD mice. Clinical (weight and food intake) and biochemical parameters (very long-chain fatty acids, abnormal bile acids, etc.) were in accordance with ZSD phenotype but they were not robustly modified by HT. As expected, one third of the infused cells were detected in the liver 24 h post-HT. No liver nor spleen microchimerism was detected after 7, 14 and 30 days. Future optimizations are required to improve hepatocyte engraftment in Pex1-G844D NMRI mouse liver. The mouse model exhibited the robustness required for ZSD liver-targeted therapies evaluation.

Robust Pre-Clinical Results and Large-Scale Manufacturing Process for Edit-301: An Autologous Cell Therapy for the Potential Treatment of SCD

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. As fetal hemoglobin (HbF) has been shown to be protective against clinical manifestation of SCD, we are developing EDIT-301, an autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a ribonucleoprotein (RNP) to promote HbF expression to treat SCD. Fetal hemoglobin induction for EDIT-301 is achieved by disrupting the HBG1 and HBG2 promoter distal CCAAT-box region where naturally occurring mutations are found to be associated with elevated HbF expression. Cas12a was selected over Cas9 due to the more productive and sustainable (NHEJ derived) indel profile, as well as high specificity. Using Cas12a RNP, on-target editing of ~90% was achieved in mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from both healthy and SCD donors at research scale with no detectable off targets. Editing of CD34+ cells led to an average of 43% and 54% of HbF expression in the erythroid progeny of normal donor and SCD donor cells respectively in a pancellular fashion (~93% population). The robust HbF induction in SCD red blood cells (RBCs) resulted in significant phenotypic and functional improvement including reduced sickling and increased deformability under hypoxia ex vivo. Using a microfluidic assay that replicated blood flow in microvasculature under varying oxygen conditions, SCD RBCs derived from RNP electroporated CD34+ cells showed improved rheological behavior. The rheology improvement under hypoxia was strongly correlated with the increased levels of HbF in each sample. Infusion of the modified CD34+ cells from normal donors into NBSGW mice resulted in long-term multi lineage and polyclonal reconstitution. Editing levels at 16 weeks post infusion were &amp;gt; 90% in all human lineages tested, demonstrating the efficient editing of SCID-repopulating hematopoietic stem cells (HSCs). Consistent with the high editing levels, human erythroid cells from the bone marrow of mice that received Cas12a-RNP treated cells demonstrated pancellular (~90% F+ RBCs) HbF expression averaging 40-50% of total hemoglobin compared to ~5% HbF observed in the control group. We have developed a consistent large-scale process using functionally closed, semi-automated systems suitable for use in clinical manufacturing. We have shown robust editing of normal donor CD34+ cells and SCID-repopulating HSCs with the clinical scale process. Editing levels of &amp;gt;90% were detected after long term engraftment in mice. In summary, we have demonstrated successful on-target editing of mPB CD34+ cells derived from both normal and SCD donors using a Cas12a RNP, which coincided with robust HbF induction and a phenotypic reduction of sickling in the SCD erythroid progeny, as well as improved rheological behavior. Editing of the HBG1 and HBG2 promoters using this RNP was highly specific with no measurable off-target. In vivo, cells from normal donors readily engrafted and reconstituted all blood cell lineages at levels comparable to unedited cells. Finally, a robust large-scale manufacturing process has been developed to supply material for the clinical setting. Based on these results, we are completing the activities required to assess EDIT-301 in the clinic as treatment for SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Heath:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Sousa:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Janoudi:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. An:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Albright:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Teixeira:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Monesmith:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Chang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company.

Effect of Nicotinamide, 1-Methylnicotinamide, and N’-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p&amp;lt;0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p&amp;lt;0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

LONG-TERM ENGRAFTMENT AND SAFETY OF HUMAN BONE MARROW DERIVED CD133+ CELLS IN A RAT MODEL OF ASHERMAN’S SYNDROME

Asherman Syndrome (AS) is defined by presence of intrauterine adhesions, due to the loss of endometrial stem cells. CD133+ bone marrow-derived stem cells (BMDSCs) contribute to human endometrium regeneration and represent an advanced cell therapy to treat this disease. Our aim is to describe the short-term and long term engraftment as well as safety and Mechanisms of Action (MoA) of pure and stable population of human CD133+ cells in a model of AS with athymic rats.

Characteristics of cells with engraftment capacity within CD34+ cell population upon G-CSF and Plerixafor mobilization.

In the context of hematopoietic cell transplantation, hematopoietic stem cells and progenitor cells (HSC and HPC) are usually collected by apheresis following their mobilization by G-CSF alone or in combination with Plerixafor® when patients fail to respond to G-CSF alone. In medical practice, the quality of the hematopoietic graft is based on CD34+ cell content that is used to define "Good Mobilizer (GM)" or "Poor Mobilizer (PM)" patients but does not report the real HSC content of grafts. In this study, we assessed the HSC content within the CD34+ fraction of graft samples from 3 groups of patients: 1-GM patients receiving G-CSF only (GMG-CSF), 2-PM patients receiving G-CSF only (PMG-CSF), 3-PM patients receiving G-CSF + Plerixafor (PMG-CSF+P). Although HSC from the 3 groups of patients displayed very similar phenotypic profiles, expression of "stemness" genes and metabolic characteristics, their capacity to engraft NSG mice differed revealing differences in terms of HSC between groups. Indeed according to mobilization regimen, we observed differences in migration capacity of HSC, as well as differences in engraftment intensity depending on the initial pathology (myeloma versus lymphoma) of patients. This suggests that mobilization regimen could strongly influence the long term engraftment efficiency of hematopoietic grafts.

Successful hematopoietic stem cell transplantation in a patient with a novel mutation in coronin 1A

Introduction: Coronin 1A is part of a family of highly conserved actin regulatory proteins with key roles in T cell homeostasis and T cell receptor signaling. Null mutations in coronin 1A result in severe combined immunodeficiency, whereas hypomorphic mutations have been associated with a somewhat milder immunological phenotype. Nevertheless, all patients described so far have markedly reduced naïve peripheral T cells, impaired T cell responses to mitogens, and limited T cell receptor diversity. Interestingly, despite poor thymic output, thymus architecture appears normal. To date, only 2 cases of hematopoietic stem cell transplantation (HSCT) have been reported in coronin 1A deficiency. Aim: To describe the identification, transplantation course, and long term outcome of a Canadian Inuit patient diagnosed with coronin 1A deficiency. Methods: Patient chart review was performed in accordance with institutional research ethics approval. A combination of immunological investigations and molecular genetic analyses were utilized to identify a novel mutation in the tryptophan-aspartate repeat region of coronin 1A. Based on the patient’s profound T cell dysfunction, the decision was made to proceed with HSCT. Results: The patient presented with a history of recurrent urinary tract infections, otitis media, and developmental delay involving poor axial and peripheral muscle tone. Axillary lymphadenopathy was noted and subsequent thymus biopsy revealed aberrant CD7+ T cell deficiency. Lymphocyte responses to mitogens and T cell receptor excision circle levels were markedly reduced, consistent with the diagnosis of severe combined immunodeficiency. Whole exome sequencing and Sanger confirmation revealed a novel mutation in coronin 1A. HSCT using a HLA-matched unrelated donor resulted in long term engraftment and solid immune reconstitution. Conclusion: Very few patients with coronin 1A deficiency have been described to date, making it difficult to evaluate its natural history and management. Here, we describe the presentation, identification, transplantation, and outcome in our patient. Statement of novelty: We describe the successful hematopoietic stem cell transplantation course and outcome in a patient with a novel mutation in coronin 1A.

Fc engineered anti-CD33mAb potentiates cytotoxicity of mbIL-21 expanded NK-cells against primary AML pre-treated with decitabine

Background & Aim Natural killer (NK) cells are limited in number and require cytokines for in vivo expansion. Recipient NK cells are shown to have both qualitative and quantitative defects in acute myeloid leukemia (AML) patients, providing rationale for infusion of donor-derived NK cells. We showed that decitabine (DAC) enhances expression of NKG2D ligands in AML blasts and renders the blasts susceptible to killing by NK cells (Vasu et al. 2016). We conducted a phase I trial giving 5-day DAC in relapsed, refractory AML patients followed by haploidentical NK cells on day 0 and six doses of IL-2, where we demonstrated short term engraftment of donor-derived NK cells (up to 48 hours) can be achieved by fludarabine and DAC. Here, we report preclinical studies using membrane bound IL-21 (mbIL-21) expanded NK cells, which can be given without extraneous cytokine support. Methods, Results & Conclusion NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying mbIL-21 and 4-1BBL (CSTX002) to derive mbIL-21 NK cells. These cells have minimal senescence despite robust proliferation with the feasibility to infuse multiple NK cell doses of 1 × 108/kg (Denman et al. 2012). We investigated if mbIL-21 NK cells exhibit natural killer activity alone or in the presence of Fc-engineered anti-CD33 antibody [antibody-dependent cellular cytotoxicity (ADCC)] against primary AML samples from patients who received DAC, pre-NK cell infusion (day-1) and at 24 days post haplo-NK infusion (Post-NK infusion). Upon incubation with primary AML blasts, mbIL-21 NK cells displayed an activation profile as evidenced by surface CD107a induction (p=0.0051) and variable donor-dependent intra-cellular IFNγ production, which was further increased with CD33mAb coated AML cells (p=0.0018). ADCC assays using chromium-51 (Cr51) labelled primary AML blasts revealed mbIL-21 NK cells lysed primary AML blasts alone which was significantly enhanced with CD33mAb coated target cells (p=0.0004). Importantly, CD33mAb dependent enhanced-cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post DAC treatment. Further, in-vivo infusion of mbIL-21 NK cells in AML patient derived xenograft (AML PDX) mice, treated with CD33mAb, reduced the tumor burden. These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of Fc engineered anti-CD33 antibody in AML.

Mgta-456 Contains Large Numbers of CD34+CD90+ Hematopoietic Stem Cells (HSC) Which Contain the NSG Engraftment Activity and Correlate with Time to Neutrophil Recovery Following Transplant into Patients with Hematologic Malignancy

Background Patient access to well-matched cord blood (CB) containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and increased risk of graft failure. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. To date, 41 patients with hematological malignancies (n=36) and non-malignant diseases (n=5) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (80 to 203 days), respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p Results After the selected CD34+ cells are cultured, the expanded product is ∼35% CD34+ and 65% CD34- (Figure 1). The CD34+ cells can be further fractionated into CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded product contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present ( Conclusion In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large numbers of CD34+CD90+ HSC, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically.

Placebo Controlled Functional Analysis of Eltrombopag in a Preclinical Xenograft Model of Myelodysplastic Syndromes (MDS)

Introduction: Thrombocytopenia is a common complication among MDS patients. Thus, many patients are dependent on platelet (PLT) transfusions, which give short-term therapeutic relief but are also associated with considerable clinical risks. In this context, thrombopoietin receptor agonists (TRAs) are under investigation as alternative treatment option, albeit with the concern that these substances may promote adverse events in MDS. However, beside potential positive effects on thrombopoiesis in MDS patients the TRA Eltrombopag (EPAG) has also been shown to exert positive disease modifying effects in vitro (Roth et al., Blood 2012). Using a MDS xenograft model, we here investigate the efficacy of EPAG and its influence on clonal composition on primary patient derived MDS xenografts and present data from an ongoing study.Methods: Currently, samples from n=18 MDS patients (MDS del(5q)=2, MDS-MLD=6, MDS-RS-MLD=1 MDS-EB-1=2, MDS-EB-2=7) have been xenografted into NSG mice by intrafemoral co-injection of CD34+ hematopoietic stem cells and mesenchymal stromal cells using a modified protocol according to Medyouf et al., Cell Stem Cell 2014. Long term engraftment is assessed 12 weeks post-transplant by intrafemoral bone marrow (BM) biopsy and mice with positive human engraftment are subsequently treated with either EPAG (50mg/kg) or vehicle control for 18 weeks. During that time, the mice are bled every two weeks and BM aspiration is performed every six weeks. Human hematopoietic cells are FACS sorted. In peripheral blood, human PLTs are specifically and absolutely counted with a FACS assay based on hCD41+ cells and beads. To track clonal composition of MDS samples upon xenografting and EPAG treatment in comparison to placebo control, the original patient sample and the final MDS xenograft sample are being whole exome sequenced (WES). Interspersed time points are analyzed with a patient individual amplicon based deep sequencing approach (Mossner et al., Blood 2016) to calculate dynamics of variant allele frequencies (VAF) in dependency of treatment.Results: To date, n=12 patient samples have been analyzed for human engraftment after 12 weeks post-transplant. Of these, n=7 (58%) have shown positive human engraftment and are being treated with EPAG versus placebo. To this end, one case has been completely followed up, including final molecular analysis. This MDS high risk case (MDS-EB-2) with a clinical PLT count of 29x109 PLT/L was transplanted into n=3 NSG mice. While two mice treated with EPAG survived the complete duration of the experiment, the placebo mouse died prematurely due to severe weight loss after 6 weeks of treatment. Further, EPAG treatment led to an initial rise of human PLT levels, while the placebo treated mouse presented a continuous decline of human PLTs, showing the efficacy of EPAG on human xenografts in the model. This observation has been confirmed in another case currently still under treatment. Molecular tracking by WES confirmed MDS patient specific molecular lesions in the MDS xenograft such as monosomy 7 and the disease related mutations CBL, DNMT3A and EZH2 with VAFs of 83%/43%/23% respectively. The monosomy 7 was detectable in all mice. CBL and DNMT3A exhibited similar VAFs in mouse EPAG1 (VAF=100%/54%), EPAG2 (VAF=100%/34%) and placebo (VAF=100%/50%). The EZH2 mutation was only detected in mouse EPAG2 (VAF=11%). Interestingly, the placebo mouse acquired a de novo mutation of U2AF1 (VAF=10%), which was not detectable in the initial patient sample or the EPAG treated mice. This spliceosomal mutation is associated with a higher risk of transformation to AML and shorter survival (Graubert et al., Nat Genet 2012; Makishima et al., Blood 2012).Conclusions: Our data show first proof of principle results that new treatment options can be tested successfully in a preclinical murine xenograft model of primary MDS patient samples in a placebo controlled experimental setting. This approach allows the performance of patient individual substance testing that can segregate substance specific effects from natural disease progression in the same patient. Clinical parameters such as human PLT production and molecular clonal composition can be measured with a high confidence in vivo. Our current data show preliminary support for the hypothesis that EPAG may be efficacious in increasing PLT production in MDS patients without adversely influencing the underlying clonal composition. DisclosuresNowak:Novartis: Research Funding.

Mgta-456 Contains Large Numbers of Expanded Cord Blood (CB) CD34+CD90+ Hematopoietic Stem Cells (HSC) Which Confer All Engraftment Activity and Correlate with Accelerated Neutrophil Recovery after Myeloablative Conditioning in Patients with Hematologic Malignancy

Background: Patient access to well-matched CB containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and a risk of graft failure relative to other hematopoietic stem cell (HSC) sources. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. Thus far, 40 patients with hematological malignancy (n=36) and non-malignant diseases (n=4) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (20 to 143 days) respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p<0.01). The aim of the current study was to fully characterize the expanded CD34+ cell fraction of MGTA-456 phenotypically and functionally and identify the cell population that correlates with time to neutrophil recovery. We found that the expanded CD34+CD90+ population of MGTA-456 were the cells responsible for engraftment in NOD-scid IL2Rgammanull (NSG) mice. We hypothesized that the dose of CD34+CD90+ cells/kg would have the strongest correlation with time to neutrophil recovery.Methods: The expansion culture consisted of StemSpan SFEM supplemented with SCF, FLT- 3L, TPO and IL-6 (all at 50 ng/mL) and the AHRa without the addition of antibiotics. After a 10 or 15 day culture, the CD34 expanded product was characterized by cell surface markers (CD34, CD90, CD133, CD41, CD71, CD235a, CD3, CD4, CD8, CD14, CD15, CD16, CD11b, CD33, CD19, CD56, and CD10), as well as colony-forming unit (CFU) capacity and engraftment potential in NOD-scid IL2Rgammanull (NSG) mice in a subset of products.Results: The expanded CD34+ cell fraction of MGTA-456 consisted of CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded fraction contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present (<1%) in the CD34 expanded fraction of MGTA-456. To identify which of these cell populations contain the NSG engraftment activity, we sorted CD34-, CD34+CD90- and CD34+CD90+ cells from MGTA-456 and transplanted the cell fractions into NSG mice. These studies clearly demonstrated that all NSG engraftment activity resided in the CD34+CD90+ cell subpopulation (Figure 2). Based on this result, we correlated the dose of CD34-, CD34+CD90-, CD34+CD90+ cells, and CFU cells/kg infused with time to neutrophil recovery of patients treated with MGTA-456. The dose of TNC (r2=0.49, P<0.05), CD34+CD90- (r2=0.45, p<0.05), and CD34+CD90+ (r2=0.52, p<0.05) cells/kg all significantly correlated with time to neutrophil recovery. The dose of CD34+CD90+ cells/kg had the strongest correlation with time to neutrophil recovery consistent with the NSG engraftment result.Conclusion: In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large doses of CD34+CD90+ HSC and progenitors, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically. [Display omitted] DisclosuresBoitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.

Abstract 5665: NOD-scid IL2rg null (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC

Abstract The study of human immune system-tumor interactions has been limited historically by a paucity of models that fully reproduce human immune system function. Humanized mice are emerging as an exciting platform to study human immuno-oncology and provide preclinical tools to test cutting edge immunotherapies. An effective and simplistic approach to engraft immunodeficient mice with functional human T cells is the injection of human peripheral blood mononuclear cells (PBMC). This PBMC engraftment model (termed Hu-PBL-SCID mice) has supported studies of human infectious agents, allograft rejection, and human T cell immune function. The Hu-PBL-SCID model has also supported studies of human immune system-tumor interactions following injection of human PBMC into immunodeficient mice implanted with patient-derived (PDX) tumors. However, a major limitation of this model is the development of acute xenogeneic graft-versus-host disease (GVHD) due to human T cell recognition of murine MHC class I and class II. To address this limitation, we created two strains of NSG mice that lack murine MHC antigens, one by crossing NSG-B2Mnull with NSG-(IA IEnull) mice, the second by knocking out the MHC class II IAb gene in NSG-(Kb Db)null mice using TALEN technology. We observed that human IgG clearance was rapid in NSG-B2Mnull (IA IEnull) mice that lack functional FcRn activity whereas clearance in NSG-(Kb Db)null (IAnull) mice was comparable to that observed in NSG mice. Injection of human PBMC into both strains led to engraftment of human T cells that exhibited an approximate 3:1 CD4:CD8 ratio with long term engraftment and without development of acute GVHD. The engrafted human T cells were functional as documented by their ability to reject human islet allografts. Administration of human recombinant IL2 using AAV-IL2 to NSG-(Kb Db)null (IAnull) mice resulted in increased human CD45 cell engraftment, including heightened levels of human Tregs. However, high IL2 levels led to the development of acute GVHD. Moreover, NSG-(Kb Db)null (IAnull) mice injected with a PDX tumor and PBMC from an allogeneic donor supported significant tumor growth in the absence of acute GVHD. These data document that NSG mice deficient in murine MHC class I and class II can support studies of human immune system-tumor interactions in the absence of acute GVHD and provide a model for evaluation of human antibody therapeutics. Citation Format: Michael A. Brehm, Michael Wiles, Laurie Kenney, Ben Low, Roland M. Tisch, Lisa Burzenski, James G. Keck, Dale L. Greiner, Leonard D. Shultz. NOD-scid IL2rgnull (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5665.

NOD-scid IL2rg null (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC

Abstract Humanized mice are emerging as an exciting platform to study human immuno-oncology and provide preclinical tools to test cutting edge immunotherapies. An effective and simplistic approach to engraft immunodeficient mice with functional human T cells is the injection of human peripheral blood mononuclear cells (PBMC). This PBMC engraftment model (termed Hu-PBL-SCID) has supported studies of human immune system-tumor interactions following injection of human PBMC into immunodeficient mice implanted with patient-derived (PDX) tumors. However, a major limitation of this model is the development of acute xenogeneic GVHD due to human T cell recognition of murine MHC class I and class II. To address this limitation, we created two strains of NSG mice that lack murine MHC antigens, one by crossing NSG-B2Mnullwith NSG-(IA IEnull) mice, the second by knocking out the MHC class II IAb gene in NSG-(KbDb)null mice using TALEN technology. We observed that human IgG clearance was rapid in NSG-B2Mnull(IA IEnull) mice that lack functional FcRn activity whereas clearance in NSG-(KbDb)null(IAnull) mice was comparable to that observed in NSG mice. Injection of human PBMC into both strains led to long term engraftment of human T cells without development of acute GVHD. The engrafted human T cells were functional as documented by their ability to reject human islet allografts. Moreover, NSG-(KbDb)null(IAnull) mice injected with a PDX tumor and PBMC from an allogeneic donor supported significant tumor growth in the absence of acute GVHD. These data document that NSG mice deficient in murine MHC class I and class II support studies of human immune system-tumor interactions in the absence of acute GVHD and provide a model to evaluate human antibody therapeutics.

Transcriptional regulation of Hhex in hematopoiesis and hematopoietic stem cell ontogeny

Hematopoietic stem cells (HSCs) emerge during development via an endothelial-to-hematopoietic transition from hemogenic endothelium of the dorsal aorta (DA). Using in situ hybridization and analysis of a knock-in RedStar reporter, we show that the transcriptional regulator Hhex is expressed in endothelium of the dorsal aorta (DA) and in clusters of putative HSCs as they are specified during murine development. We exploited this observation, using the Hhex locus to define cis regulatory elements, enhancers and interacting transcription factors that are both necessary and sufficient to support gene expression in the emerging HSC. We identify an evolutionarily conserved non-coding region (ECR) in the Hhex locus with the capacity to bind the hematopoietic-affiliated transcriptional regulators Gata2, SCL, Fli1, Pu.1 and Ets1/2. This region is sufficient to drive the expression of a transgenic GFP reporter in the DA endothelium and intra-aortic hematopoietic clusters. GFP-positive AGM cells co-expressed HSC-associated markers c-Kit, CD34, VE-Cadherin, and CD45, and were capable of multipotential differentiation and long term engraftment when transplanted into myelo-ablated recipients. The Hhex ECR was also sufficient to drive expression at additional blood sites including the yolk sac blood islands, fetal liver, vitelline and umbilical arteries and the adult bone marrow, suggesting a common mechanism for Hhex regulation throughout ontogenesis of the blood system. To explore the physiological requirement for the Hhex ECR region during hematoendothelial development, we deleted the ECR element from the endogenous locus in the context of a targeted Hhex-RedStar reporter allele. Results indicate a specific requirement for the ECR in blood-associated Hhex expression during development and further demonstrate a requirement for this region in the adult HSC compartment. Taken together, our results identified the ECR region as an enhancer both necessary and sufficient for gene expression in HSC development and homeostasis. The Hhex ECR thus appears to be a core node for the convergence of the transcription factor network that governs the emergence of HSCs.

Poor Mobilizing Lymphoma Patients Rescued By Addition of Plerixafor during Stem Cell Mobilization Have Equal Survival As Good Mobilizers When Given High Dose Therapy with Autologous Stem Cell Support

High dose therapy (HDT) followed by autologous stem cell support is a curative treatment option in selected lymphoma patients. To obtain a quick and sustained engraftment after high dose therapy, a minimal number of 2x106 CD34+ cells/kg are preferable. However, in 15-20% of the lymphoma patients insufficient numbers of CD34+ cells are harvested, characterized as poor mobilizers. Furthermore, previous findings have shown that poor mobilizing lymphoma patients given HDT have a less favorable prognosis than good mobilizers. In recent years, new mobilizing agents including plerixafor, a CXCR4 antagonist, have been developed. Our previous findings have shown that with a low concentration of CD34+ cells (4-10 cells/µL) and a total white cell count >4x109/L, addition of plerixafor resulted in successful stem cell harvesting in these poor mobilizing patients.In the present study, we have mobilized 37 poor-mobilizing lymphoma patients with addition of plerixafor from January 2010 to December 2014. Four patients were mobilized with G-CSF and plerixafor, whereas 33 patients were given plerixafor in addition to chemotherapy and G-CSF. Thirty four patients underwent a successful stem cell harvest and received high dose therapy. The patients were followed up after reinfusion of autologous stem cells with regard to short term and long term engraftment as well as relapse and death.The day prior to harvest, the level of leukocytes was 12,6x109 cells/L (median; range3,3-65,2), and the CD34+ concentration was 5,7x106/L (median; range 1,9-12,4). Following plerixafor injection, the concentration of CD34+ cells increased to 23,4 x106/L (median; range 5,6-65,6). The patients were then harvested (median: 3,9x106 CD34+cells/kg; range: 1,5-7,3) with 1-2 days of apheresis, One of the patients received high dose therapy although we only obtained 1.8x106 CD34+cells/kg at stem cell harvest.In the 34 lymphoma patients receiving high dose therapy followed by autologous stem cell support we observed that time to short term engraftment, defined by neutrophils >0.5x109/L and thrombocytes >20x109/L were 12 days (median; range 8-19) and 14 days (median; range 10-100) respectively. Furthermore, as an estimate of long term engraftment we examined the thrombocyte levels at day 100 after reinfusion. The median level was 132x109/L (range 27-398). Moreover, in contrast to previous findings we have observed durable responses with event free survival of 56% and overall survival of 67% (Median observation time without relapse: 19,5 months, range 0-64 months).In conclusion, our findings show that addition of plerixafor is successful in mobilization of hematopoietic stem cells in poor mobilizing patients resulting in a quick and sustained engraftment as well as durable responses and similar prognosis as for good mobilizing lymphoma patients after high dose therapy with autologous stem cell support. DisclosuresNo relevant conflicts of interest to declare.