Endothelial cell-derived angiopoietin-like protein 2 supports hematopoietic stem cell activities in bone marrow niches

Bone marrow niche cells have been reported to fine-tune HSC stemness via direct interaction or secreted components. Nevertheless, how niche cells control HSC activities remains largely unknown. We previously showed that angiopoietin-like protein 2 (ANGPTL2) can support the ex vivo expansion of HSCs by binding to human leukocyte immunoglobulin-like receptor B2 (LILRB2). However, how ANGPTL2 from specific niche cell types regulates HSC activities under physiological conditions is still not clear. Herein, we generated an Angptl2-flox/flox transgenic mouse line and conditionally deleted Angptl2 expression in several niche cells, including Cdh5+ or Tie2+ endothelial cells, Prx1+ mesenchymal stem cells and Pf4+ megakaryocytes, to evaluate its role in the regulation of HSC fate. Interestingly, we demonstrated that only endothelial cell-derived ANGPTL2 and not ANGPTL2 from other niche cell types plays important roles in supporting repopulation capacity, quiescent status and niche localization. Mechanistically, ANGPTL2 enhances PPARD expression to transactivate G0s2 to sustain the perinuclear localization of nucleolin to prevent HSCs from entering the cell cycle. These findings reveal that endothelial cell-derived ANGPTL2 serves as a critical niche component to maintain HSC stemness, which may benefit the understanding of stem cell biology in bone marrow niches and the development of a unique strategy for the ex vivo expansion of HSCs.

A Novel Method for Highly Efficient Ex Vivo Expansion and Genetic Engineering of Human Natural Killer Cells for Cancer Therapy

Cancer immunotherapy with adoptive transfer of human leukocyte antigen-mismatched, CD19-targetd chimeric antigen receptor (CAR)-transduced natural killer (NK) cells has attracted attention because of its efficacy and safety when infused in patients with refractory and relapsed B-cell lymphomas. However, generating clinical doses of CAR-NK cells is still a challenge. The methods for ex vivo expansion and genetic modification of primary human NK cells usually rely on the use of irradiated feeder cell lines, which has been restrictive due to high costs, scale-up difficulties, and licensing restrictions. Hence, novel strategies that do not require feeder cells will be beneficial in standardizing these types of cell therapies. In this study, we show the effectiveness of a novel feeder-free culture system in expanding NK cells ex vivo and generating CAR-NK cells. Unsorted peripheral blood mononuclear cells (PBMCs) collected from healthy donors were cultured with a reagent containing dissolvable microspheres that are conjugated with anti-CD2/NKp46 antibodies (Cloudz TM Human NK Cell Expansion Kit) and a combination of multiple cytokines, including interleukin (IL)-2, IL-12, IL-18, and IL-21 in medium supplemented with 10% fetal bovine serum. The activated NK cells were transduced using the RD114-pseudotyped retrovirus vector. To test whether the reagent promoted ex vivo NK cell expansion, we cultured PBMCs from 13 donors. The percentage of NK (CD56 + CD3 -) cells of initially isolated PBMCs was 15.3±7.5%. In the presence of multiple cytokine combinations, NK cell purity gradually increased and reached 91.6±7.6% by day 21. The NK cells expanded to 75.6±59.2-fold at day 10, 334±217-fold at day 14 and 1,542±913-fold at day 21. The expanded NK cells degranulated and produced intracellular cytokines upon exposure to K562 myeloid leukemia cells. The NK cells efficiently killed myeloid leukemia cells, such as K562, THP1, and KG1. The expression pattern of killer cell immunoglobulin-like receptors on NK cells remained unchanged. The expression of activating NK cell receptors, including NKp30 and NKp44, increased after 21 days of culture. Thereafter, a gene transfer to the primary human NK cells was conducted. We tested transduction efficiency and yields of modified cells on 7 days after the procedure by empty-vector transduction into NK cells expanded ex vivo for 3, 6, and 10 days (n=3, each). The results were presented as means ± standard deviation; 55.6±11.6%, 61.6±14.1%, and 73.6%±6.2% for GFP positivity in NK cells and 5.7±1.5 folds, 56.4±42.2 folds, and 12.7±5.8 folds for yields of modified cells. We selected the condition in which the transduction was carried out using NK cells expanded ex vivo for 6 days, although the differences were not statistically significant. Next, anti-CD19 CAR with a 4-1BB costimulatory and CD3z domain was transduced into NK cells. We confirmed high transduction efficiency (59.8% ±20.5%, n=3) and high CAR protein expression on the cell surface, while NK cells maintained their purity and minimal T cell outgrowth was observed. CAR-NK cells maintained their proliferative status and further expanded 15.2±6.4-fold after 7 days of the procedure. To determine whether the generated anti-CD19 CAR-NK cells had a specific effect on B cell malignancies, a CD107a mobilization assay, intracellular cytokine assay, and a flow cytometry-based cytotoxicity assay was employed. We found that CAR transduction could render NK cells to generate specific and powerful responses against CD19-positive, NK-resistant leukemia and lymphoma cell lines, such as BCR-ABL-positive acute lymphoblastic leukemia (ALL) OP-1, Burkitt lymphoma Raji, and KMT2A-rearranged ALL RS4;11, at various effector: target (ET) ratios. For example, in a 4 hour -assay, the cytotoxic effects of anti-CD19 CAR-NK cells showed 86.9±0.2% cytotoxicity against OP-1, while mock NK cells showed 17.4±2.9% cytotoxicity (ET ratio 1:1). In conclusion, this study revealed highly efficient functions of the novel feeder-free culture system, including highly efficient ex vivo expansion of primary human NK cells and generation of genetically modified NK cells for cancer immunotherapy. In future studies, we will investigate large-scale cultures using specialized flasks and GMP-grade reagents for clinical translation and the in vivo activities of the cell products in mouse xenograft models. Disclosures Imai: Juno Therapeutics: Patents & Royalties: chimeric receptor with 4-1BB signaling domain.

373 Enhancement of TCR-engineered T-cells targeting MAGE-A4 antigen by co-expression of CD8α and inhibition of AKT signaling during ex vivo T-cell expansion

BackgroundAutologous Specific Peptide Enhanced Affinity Receptor (SPEAR) T-cells targeting MAGE-A4 can be effective treatment for solid tumors.1–3 To improve efficacy, we developed a next-generation SPEAR-T cell targeting MAGE-A4 co-expressing CD8α (ADP-A2M4CD8). ADP-A2M4CD8 is under investigation in the Phase 1 SURPASS trial (NCT04044859). Enhancements have also been made to the manufacturing process with an AKT inhibitor (AKTi) during ex vivo expansion to provide a greater proliferative potential and enhanced memory phenotype.4MethodsSPEAR-T cells were manufactured using a Lentiviral vector with CD8α and MAGE-A4 targeted TCR genes. AKTi was added during ex vivo expansion. T-cell attributes were evaluated, including markers of differentiation (flow cytometry), capacity for in vitro tumor lysis (Incucyte) and changes to gene expression (scRNASeq) initially assessed with the first-gen product. Post-infusion, the presence of transduced T-cells in the peripheral circulation (PCR) and levels of inflammatory cytokines in serum (MesoScale Discovery Assay [MSD]) were evaluated.ResultsAs of May 24, 2021, 18 patients with 9 different primary tumor types were evaluable. Twelve pts received product that had AKTi during manufacture. Five patients had objective responses (RECIST), and 10 had stable disease. Responses occurred at lower MAGE-A4 expression levels and lower transduced T-cell doses relative to the first-gen product targeting MAGE-A4.1 CD4 T-cells from manufactured ADP-A2M4CD8 demonstrated direct in vitro tumor cell killing similar to CD8+ T-cells (Incucyte). scRNASeq gene expression profiles of first-gen ADP-A2M4 product manufactured with AKTi revealed the AKTi-expanded T-cells had a greater proliferation or an enhanced memory phenotype; scRNASeq analyses are ongoing for the ADP-A2M4CD8 product.An increase in IL-12 levels (MSD) in serum post-infusion suggests that endogenous immune cells are being activated, further resulting in increased levels of IFN gamma (MSD) secretion relative to patients who received first-gen product. Manufacturing with AKTi resulted in T-cells with a less differentiated phenotype (flow cytometry), and post-infusion was associated with enhanced antigen-specific serum cytokine responses, increased proliferative state (i.e., elevated levels of IL-2), and higher persistence of T-cells in peripheral blood by PCR.ConclusionsSPEAR T-cells targeting MAGE-A4 expressing cancers have been enhanced by co-expressing CD8α and adding AKTi during manufacture. These enhanced products improve CD4+ T-cell killing, release more inflammatory cytokines, proliferate more robustly with an early memory phenotype, and better engage the patient‘s endogenous immune system when compared to first-gen products or next-gen manufactured without AKTi.Trial RegistrationNCT04044859ReferencesHong, et al. ASCO 2020.D’Angelo, et al. ASCO 2021.Hong, et al. SITC 2020.Mardilovich, et al. SITC 2020.