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Lentiviral vectors (LVs) have recently witnessed an increasing demand in research and clinical applications. Their current purification processes represent the main bottleneck in their widespread use, as the methods used are cumbersome and yield low recoveries. We aimed to develop a one-step method to specifically purify LVs, with high yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging HEK293T cells were genetically engineered with a cyclical biotin-mimicking peptide displayed on a CD8α stalk, termed cTag8. LVs were modified with cTag8 by its passive incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. Expression of cTag8 on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As cTag8 binds streptavidin in the nanomolar range, the addition of micromolar concentrations of biotin resulted in the release of captured LVs by competitive elution, with overall yields of ≥60%. Analysis of eluted LVs revealed high purity with a >3-log and 2-log reduction in DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using monolith affinity chromatography, with an encouraging preliminary overall yield of 20%. This method will be of valuable use for both research and clinical applications of LVs. Keywords: lentiviral vectors, synthetic peptide, biotin mimic, streptavidin, purification, affinity chromatography, competitive elution

High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA cluster for leukemia maintenance has not been fully explored. Primary leukemias were generated in hematopoietic stem/progenitor cells from Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation and colony formation in which surviving leukemic cells retained at least one copy of the Hoxa cluster, indicating dependency. Comparative transcriptome analysis of Hoxa wild type and deleted leukemic cells identified a unique gene signature associated with key pathways including transcriptional mis-regulation in cancer, the Fanconi anemia pathway and cell cycle progression. Further bioinformatics analysis of the gene signature identified a number of candidate FDA-approved drugs for potential repurposing in high HOXA expressing cancers including MLLr leukemias. Together these findings support dependency for an MLLr leukemia on Hoxa expression and identified candidate drugs for further therapeutic evaluation.

Despite the success of autologous anti-CD19 CAR T cell therapy in B-Acute lymphoblastic leukaemia (B-ALL) and Diffuse Large B Cell Lymphoma (DLBCL), treatment failures occur. One contributing factor may be the intrinsic T cell fitness of the CAR T cell product that is influenced by the underlying malignancy and prior treatments. With the advent of gene editing, 'off the shelf' non-HLA matched healthy donor (HD) CAR T cells are under investigation for the treatment of patients (pts) in clinical trials. UCART19 (S68587) is a first-in-class allogeneic CAR T cell product expressing a second generation anti-CD19 CAR with TALEN®-mediated gene knockouts of T cell receptor alpha chain (TRAC) and CD52 to prevent graft versus host disease and to render them resistant to anti-CD52 antibody used for lymphodepletion. Preliminary clinical trial data on the use of UCART19 in B-ALL was previously reported at ASH (Benjamin et al, 2018). The phenotypic and functional characteristics of CAR T cell products manufactured from B-ALL, Chronic Lymphocytic Leukaemia (CLL) and DLBCL pts were compared to young adult healthy donor (HD) CAR T cell products. In addition, potential effects related to knocking out TRAC in HD TCR-CAR T cells were examined. Thawed PBMCs from B-ALL, CLL, DLBCL pts and HDs underwent T cell enrichment, activation with anti-CD3/CD28 beads and IL-2, followed by transduction with anti-CD19 4-1BB CD3ζ lentiviral CAR construct and expansion. HD TCR- CAR T cells were manufactured by electroporation of HD CAR T cells with mRNA coding for TRAC TALEN® and residual TCRαβ+cells were removed by magnetic bead selection. CAR expression levels, T cell subsets, and exhaustion markers were examined by flow cytometry. Expression of activation markers CD25 and CD69 was measured in response to co-culture with the CD19+cell line NALM-6. Cytotoxicity against NALM-6 and Raji was assessed and antigen-mediated proliferation measured over 14 days. HD CAR T cells (n=11) expanded significantly more during manufacture than CAR T cells derived from B-ALL (n=9), CLL (n=8) or DLBCL (n=8) pts. As expected, the electroporation step resulted in a transient decrease in viability which recovered over time in culture (n=10). Median CAR expression level was higher on CLL CAR T cell products compared to those from B-ALL pts and HDs, thought to be due to a higher CD4:CD8 ratio in some CLL products. As a consequence of TCR knockout, CD3 expression was lost on HD TCR- CAR T cells (n=10), apart from a small population of γδ CAR T cells. CLL and DLBCL CD8+CAR+cells expressed higher levels of PD1 than HD CD8+CAR+cells. DLBCL CD4+CAR+cells also expressed significantly higher levels of PD1 than HD or HD TCR-CD4+CAR+T cells. CAR+CD8+CD27+PD1- T cells have been previously described as a functionally important population that correlated with clinical outcome in pts who received CLL CAR T cells (Fraietta et al, 2018). We found HD (n=13) and HD TCR- (n=10) CAR T cells had significantly more CD8+CD27+PD1- CAR T cells compared to those derived from CLL (n=8) and DLBCL (n=6) pts, but similar levels to B-ALL pts (n=10). In the absence of CD19 antigen, DLBCL CAR+CD8+ T cells (n=6) had greater expression of CD25 and CD69. However, in response to stimulation with CD19+ NALM-6 cells, HD (n=12), HD TCR- (n=10) and B-ALL (n=10) CAR T cells had higher fold increase in CD69+ cells compared to DLBCL (n=6) CAR T cells. On paired analysis (n=6), no difference was seen in activation in response to CD19 antigen on HD compared to HD TCR- CAR T cells. All CAR T cell products demonstrated comparable cytotoxicity against NALM-6 and Raji cell lines in short term in vitro assays. However, long-term cytotoxicity will be evaluated in a murine model. We performed a detailed comparison of the phenotypic and functional characteristics of CAR T cells derived from pts with B-cell malignancies and HDs. DLBCL CAR T cells showed lower antigen specific activation but higher baseline activation which could lead to more differentiated exhausted T cells. CAR T cells derived from HDs show a higher proportion of the therapeutically relevant CAR+CD8+CD27+PD1- cells compared to patients with mature B cell malignancies (CLL and DLBCL), which is maintained after TRAC knockout. This suggests allogeneic CAR T cells, such as UCART19, may provide a more effective product for pts with T cell dysfunction. Disclosures Graham: Gillead: Other: Funding to attend educational meeting; Servier: Research Funding. Jozwik:Servier: Research Funding. Metelo:Pfizer: Research Funding; Allogene: Research Funding. Almena-Carrasco:Servier: Employment. Peranzoni:Servier: Employment. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Dupouy:Servier: Employment. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Patten:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Benjamin:Amgen: Honoraria; Allogene: Research Funding; Gilead: Honoraria; Servier: Research Funding; Eusapharm: Consultancy; Pfizer: Research Funding; Takeda: Honoraria; Novartis: Honoraria.

Introduction Hox gene expression is high in hematopoietic stem/progenitor cells (HSPCs), decreases during normal differentiation but remains elevated in leukemia subtypes. Polycomb repressor complexes and histone modifiers, e.g. Mixed Lineage Leukemia (MLL), are key regulators of Hox expression. MLL rearrangements, frequent in acute leukemia, are associated high HOXA expression. However, necessity for the HoxA cluster in MLL-leukemia maintenance is not fully elucidated. Methodology Ectopic overexpression of MLL-AF9 (MA9) in HSPCs in conditional compound transgenic mouse backgrounds MxCre+/HoxAflox/flox (MAFF) or HoxAflox/flox (AFlox) models resulted in increased colony formation and growth in liquid culture. Transformed colonies, serially re-plated (n=5) in methylcellulose and transplanted into sub-lethally irradiated recipient mice, resulted in primary leukemia. Initially, MAFF-MA9 leukemias were used to examine in vivo deletion of the HoxA cluster using intraperitoneal injections of Poly(I:C) to initiate an interferon response. To further examine the necessity for the HoxA cluster in disease maintenance, AFlox-MA9 leukemias were treated ex vivo with Cre-recombinase (MSCV-Cre-GFP) or vector control (MSCV-GFP), sorted based on GFP expression and used for gDNA-PCR, gene expression (Illumina BeadArray) and transplantation into sub-lethally irradiated recipient mice (500 cGy). Results Generation of MLL-AF9 leukemias in the MAFF background (MAFF-MA9) resulted in deletion of one HoxA cluster allele (HoxA+/-), validated by genomic PCR and gDNA sequencing from expanded single colonies (Figure 1) presumably due to viral-induced activation of the Mx1 promoter. PolyI:C treatment of these mice resulted in a modest extension in survival (1-2 days) compared to controls. Luciferase labelling and transplantation of MAFF-MA9 leukemias into NSG mice, followed by PolyI:C treatment, showed a measurable decrease in disease burden compared to control, however this did not correlate with overall survival. Direct treatment of MAFF-MA9 cells with interferon-α (in vitro) resulted in further deletion of the HoxA cluster (HoxA-/hypo) and significant reduction in colony formation compared to controls. Although non-leukemic MAFF HSPCs retained colony forming ability after complete HoxA cluster deletion (HoxA-/-) no HoxA-/- colonies were recovered from the interferon-α treated MAFF-MA9 cultures. Cre-recombinase-induced deletion of the Hoxa cluster from AFlox-MA9 leukemia cells was confirmed by gDNA-PCR and sequencing (Figure 1). Transplantation of Cre-treated AFlox-MA9 cells resulted in significant increased survival (P Conclusion Together these data support a fundamental role for the HoxA cluster in MLL-AF9 maintenance indicating dependency for this leukemia subtype which may be exploited for therapeutic benefit. Figure 1. Deletion of Hoxa cluster validated by Sanger sequencing. The chromatograph (A) and sequence obtained (B) from PCR products generated from primers used to detect Hoxa cluster deletion with retention of the 5' UTR and 3' UTR regions of the Hoxa13 and Hoxa1 genes respectively. Figure 1. Deletion of Hoxa cluster validated by Sanger sequencing. The chromatograph (A) and sequence obtained (B) from PCR products generated from primers used to detect Hoxa cluster deletion with retention of the 5' UTR and 3' UTR regions of the Hoxa13 and Hoxa1 genes respectively. Figure 2. Deletion of the Hoxa cluster results in significant increase in survival of Cre-GFPHi AFlox-MA9 mice compared to GFPhi control mice (n=10). Significance as calculated by Log-rank (Mantel-Cox) Test is denoted *** = p Disclosures No relevant conflicts of interest to declare.