Improved Bone Marrow Homing of NK Cells through PM21 Particle Stimulated Enhancement of Fucosylation of E-Selectin Ligand

Introduction

Therapy of marrow borne disease such as leukemias or myelomas require the eradication of tumor cells that are resident in the bone marrow. Cell based therapies are increasingly a promising modality toward potential complete cures of such marrow malignancies. In particular, natural killer (NK) cell therapeutics are an attractive therapeutic modality due to their inherent ability to broadly detect and kill tumors. However, for NK cells to kill the marrow resident blasts, they must traffic to the bone marrow. Key for marrow homing is the proper fucosylation of extracellular proteins (such as e.g. P-selectin glycoprotein ligand-1, PSGL) on NK cells which is necessary for E-selectin binding. PM21 particles are plasma membrane particles prepared from CSTX002 cell line (K562 cells modified to express membrane bound IL-21 [mbIL21] and 4-1bbl, K562.mbIL21.41BBL) and can be used to potently stimulate expansion of NK cells that have high anti-tumor potency, similarly as to the expansion with CSTX002 feeder cells that have mbIL21 stimulation. Shown herein is that NK cells stimulated with PM21 particles have increased fucosylation of E-selectin binding ligands and have increased homing to the bone marrow in mice.

Methods

NK cells were expanded from PBMCs obtained from healthy donors by stimulating with PM21 particles (200 µg/mL) in SCGM media supplemented with 10% FBS and IL-2 (100 U/mL). PM21 particles were prepared from CSTX002 cell line as previously described. The fucosylation status of E-selectin ligands on expanded NK cells were analyzed by flow cytometry with the HECA452 mAb that only binds the fucosylated forms. ChIP-seq analysis was performed on NK cells expanded from feeder cell co-cultures with CSTX002 and by probing with anti-STAT3. For physiological trafficking studies, whole PBMCs were pre-activated in vitro for two days with PM21 particles (200 µg/mL) and then injected ip into NSG mice and treated or not with PM21-particles (2x/weekly). Organs were harvested 16 days later and analyzed by flow cytometry for the presence of human NK cells.

Results

NK cells expanded using PM21 or other stimulation methods were comparatively analyzed for fucosylation status by staining with HECA452. The NK cells stimulated with mbIL21, either through PM21 or CSTX002 feeder cells, were found to have highest HECA452 staining compared NK cells stimulated with IL-2, soluble IL-21, soluble 4-bbl, unmodified K562, or K562 modified with only mbIL21 (no 4-1bbl). To determine transcriptional changes responsible for the apparent increase in fucosylation, anti-STAT3 ChIP-seq analysis was performed on naïve NK cells or those expanded with CSTX002. STAT3 is a transcription factor that is activated upon IL-21 stimulation. The anti-STAT3 ChIP-seq analysis elucidated that CSTX002 stimulated NK cells have enhanced STAT3 binding to the FUT7 gene and that FUT7 expression increased. To determine if stimulation with PM21 particles can enhance homing of NK cells to bone marrow, NSG mice were injected ip with NK cells stimulated in vitro for 2 days with PM21, and then the mice were treated with or without PM21 particles (ip, 800 µg, 3x per week). Analysis of the bone marrow from sacrificed animals showed that a greater amount of human NK cell were present in the marrow in animals treated with ip doses of PM21 particles.

Conclusions

NK cells expanded with PM21 particles had increased fucosylation of likely E-selecting ligands. This higher degree of fucosylation is likely due to the enhanced expression of fucosyl transferase 7, shown bye ChIP-seq analysis of CSTX002 stimulated NK cells. In vivo studies show that stimulation with PM21 does allow for increased trafficking of NK cells to the bone marrow.

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